Bicyclic pyrazolo protein kinase modulators

ABSTRACT

The present invention provides novel bicyclic pyrazolo kinase modulators and methods of using the novel bicyclic pyrazolo kinase modulators to treat diseases mediated by kinase activity.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/530,111, filed Dec. 17, 2003, and U.S. Provisional Application No.60/598,221, filed Aug. 2, 2004, each of which is herein incorporated byreference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Mammalian protein kinases are important regulators of cellularfunctions. Because disfunction in protein kinase activity have beenassociated with several diseases and disorders, protein kinases aretargets for drug development.

The tyrosine kinase receptor, FMS-like tyrosine kinase 3 (FLT3), isimplicated in cancers, including leukemia, such as acute myeloidleukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplasia.About one-quarter to one-third of AML patients have FLT3 mutations thatlead to constitutive activation of the kinase and downstream signalingpathways. Although in normal humans, FLT3 is expressed mainly by normalmyeloid and lymphoid progenitor cells, FLT3 is expressed in the leukemiccells of 70-80% of patients with AML and ALL. Inhibitors that targetFLT3 have been reported to be toxic to leukemic cells expressing mutatedand/or constitutively-active FLT3. Thus, there is a need to developpotent FLT3 inhibitors that may be used to treat diseases and disorderssuch as leukemia.

The Abelson non-receptor tyrosine kinase (c-Abl) is involved in signaltransduction, via phosphorylation of its substrate proteins. In thecell, c-Abl shuttles between the cytoplasm and nucleus, and its activityis normally tightly regulated through a number of diverse mechanisms.Abl has been implicated in the control of growth-factor and integrinsignaling, cell cycle, cell differentiation and neurogenesis, apoptosis,cell adhesion, cytoskeletal structure, and response to DNA damage andoxidative stress.

The c-Abl protein contains approximately 1150 amino-acid residues,organized into a N-terminal cap region, an SH3 and an SH2 domain, atyrosine kinase domain, a nuclear localization sequence, a DNA-bindingdomain, and an actin-binding domain.

Chronic myelogenous leukemia (CML) is associated with the Philadelphiachromosomal translocation, between chromosomes 9 and 22. Thistranslocation generates an aberrant fusion between the bcr gene and thegene encoding c-Abl. The resultant Bcr-Abl fusion protein hasconstitutively active tyrosine-kinase activity. The elevated kinaseactivity is reported to be the primary causative factor of CML, and isresponsible for cellular transformation, loss of growth-factordependence, and cell proliferation.

The 2-phenylaminopyrimidine compound imatinib (also referred to asSTI-571, CGP 57148, or Gleevec) has been identified as a specific andpotent inhibitor of Bcr-Abl, as well as two other tyrosine kinases,c-kit and platelet-derived growth factor receptor. Imatinib blocks thetyrosine-kinase activity of these proteins. Imatinib has been reportedto be an effective therapeutic agent for the treatment of all stages ofCML. However, the majority of patients with advanced-stage or blastcrisis CML suffer a relapse despite continued imatinib therapy, due tothe development of resistance to the drug. Frequently, the molecularbasis for this resistance is the emergence of imatinib-resistantvariants of the kinase domain of Bcr-Abl. The most commonly observedunderlying amino-acid substitutions include Glu255Lys, Thr315Ile,Tyr293Phe, and Met351Thr.

MET was first identified as a transforming DNA rearrangement (TPR-MET)in a human osteosarcoma cell line that had been treated withN-methyl-N′-nitro-nitrosoguanidine (Cooper et al. 1984). The METreceptor tyrosine kinase (also known as hepatocyte growth factorreceptor, HGFR, MET or c-Met) and its ligand hepatocyte growth factor(“HGF”) have numerous biological activities including the stimulation ofproliferation, survival, differentiation and morphogenesis, branchingtubulogenesis, cell motility and invasive growth. Pathologically, METhas been implicated in the growth, invasion and metastasis of manydifferent forms of cancer including kidney cancer, lung cancer, ovariancancer, liver cancer and breast cancer. Somatic, activating mutations inMET have been found in human carcinoma metastases and in sporadiccancers such as papillary renal cell carcinoma. The evidence is growingthat MET is one of the long-sought oncogenes controlling progression tometastasis and therefore a very interesting target. In addition tocancer there is evidence that MET inhibition may have value in thetreatment of various indications including: Listeria invasion,Osteolysis associated with multiple myeloma, Malaria infection, diabeticretinopathies, psoriasis, and arthritis.

The tyrosine kinase RON is the receptor for the macrophage stimulatingprotein and belongs to the MET family of receptor tyrosine kinases. LikeMET, RON is implicated in growth, invasion and metastasis of severaldifferent forms of cancer including gastric cancer and bladder cancer.

Because kinases have been implicated in numerous diseases andconditions, such as cancer, there is a need to develop new and potentprotein kinase inhibitors that can be used for treatment. The presentinvention fulfills these and other needs in the art. Although certainprotein kinases are specifically named herein, the present invention isnot limited to inhibitors of these kinases, and, includes, within itsscope, inhibitors of related protein kinases, and inhibitors ofhomologous proteins.

BRIEF SUMMARY OF THE INVENTION

It has been discovered that, surprisingly, bicyclic pyrazolo compoundsmay be used to modulate kinase activity and to treat diseases mediatedby kinase activity. These novel bicyclic pyrazolo kinase modulators aredescribed in detail below. In addition, inhibitory activities ofselected compounds are disclosed herein.

In one aspect, the present invention provides a bicyclic pyrazolo kinasemodulator having the formula:

In Formula (I), X is —S—, —O—, or —N(R¹⁰)—. R¹⁰ is hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

L¹ is —C(Z)—, or —SO₂—. Z is ═O, ═S, or ═NR¹¹. R¹¹ is hydrogen, —OH,cyano, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

R¹ is hydrogen, —CF₃, amino, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl,—OR¹², or —NR¹³R¹⁴. R¹², R¹³, and R¹⁴ are independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R² is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In another aspect, the present invention provides methods of modulatingprotein kinase activity using the bicyclic pyrazolo kinase modulators ofthe present invention. The method includes contacting the protein kinasewith a bicyclic pyrazolo kinase modulator.

In another aspect, the present invention provides methods of treating adisease mediated by kinase activity in an organism. The method includesadministering to the patient a therapeutically effective amount of abicyclic pyrazolo kinase modulator of the present invention.

In another aspect, the present invention provides a pharmaceuticalcomposition including a bicyclic pyrazolo kinase modulator in admixturewith a pharmaceutically acceptable excipient.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or cyclic hydrocarbon radical, or combination thereof, which may befully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include, but are not limited to, groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by —CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will havefrom 1 to 24 carbon atoms, with those groups having 10 or fewer carbonatoms being preferred in the present invention. A “lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generallyhaving eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to two heteroatoms maybe consecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— andCH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxo,alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula C(O)OR′—represents both —C(O)OR′— and —R′OC(O)—. As described above, heteroalkylgroups, as used herein, include those groups that are attached to theremainder of the molecule through a heteroatom, such as —C(O)R′,—C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” isrecited, followed by recitations of specific heteroalkyl groups, such as—NR′R″ or the like, it will be understood that the terms heteroalkyl and—NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like.

An “alkylesteryl,” as used herein, refers to a moiety having the formulaR′—C(O)O—R″, wherein R′ is an alkylene moiety and R″ is an alkyl moiety.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

The term “cycloalkylalkyl” refers to a 3 to 7 membered cycloalkyl groupattached to the remainder of the molecule via an unsubstituted alkylenegroup. Recitation of a specific number of carbon atoms (e.g. C₁-C₁₀cycloalkylalkyl) refers to the number of carbon atoms in the alkylenegroup.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a carbon or heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent derivatives of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, naphthyloxy)propyl, and the like). However, the term“haloaryl,” as used herein is meant to cover only aryls substituted withone or more halogens.

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”, “aryl,” “heteroaryl” as well as their divalentradical derivatives) are meant to include both substituted andunsubstituted forms of the indicated radical. Preferred substituents foreach type of radical are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative radicals (including those groupsoften referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From above discussion of substituents, one of skill inart will understand that the term “alkyl” is meant to include groupsincluding carbon atoms bound to groups other than hydrogen groups, suchas haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃,—C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl radicals above,exemplary substituents for aryl and heteroaryl groups (as well as theirdivalent derivatives) are varied and are selected from, for example:halogen, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —C(O)NR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR″C(O)OR′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxo, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on aromatic ring system; andwhere R′, R″, R′″ and R″″ are preferably independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C′R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″ and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

The compounds of the present invention may exist as salts. The presentinvention includes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisometric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C— or. ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (3H), iodine-125(¹²⁵I) or carbon-14 (14C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

The terms “a,” “an,” or “a(n)”, when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

Description of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, physiological conditions.

The terms “treating” or “treatment” in reference to a particular diseaseincludes prevention of the disease.

I. Bicyclic Pyrazolo Kinase Modulators

In one aspect, the present invention provides a bicyclic pyrazolo kinasemodulator having the formula:

In Formula (I), X is —S—, —O—, or —N(R¹⁰)—. R¹⁰ is hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

L¹ is —C(Z)—, or —SO₂—. Z is ═O, ═S, or ═NR¹¹. R¹¹ is hydrogen, —OH,cyano, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

R¹ is hydrogen, —CF₃, amino, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl,—OR¹², or —NR¹³R¹⁴. R¹², R¹³, and R¹⁴ are independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R¹³ andR¹⁴ may be joined to from a ring with the nitrogen to which they areattached, wherein the ring is substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R² is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In some embodiments, R² is attached to the remainder of the molecule viaa carbon atom to form a carbon-carbon bond.

In other embodiments, if R² is unsubstituted phenyl, X is S, L₁ is—C(Z)—, and Z is ═O, then R¹ is not unsubstituted phenyl. In anotherembodiments, if R² is unsubstituted aryl, X is S, L₁ is —C(Z)—, and Z is═O, then R¹ is not unsubstituted aryl. In other embodiments, if R² issubstituted or unsubstituted phenyl, X is S, L₁ is —C(Z)—, and Z is ═O,then R¹ is not substituted or unsubstituted phenyl. In anotherembodiment, R² and R¹ are not simultaneously unsubstituted phenyl. Inanother embodiment, R² and R¹ are not simultaneously unsubstituted aryl.In another embodiment, R² and R¹ are not simultaneously substituted orunsubstituted phenyl.

In an exemplary embodiment, X is —S—. In a related embodiment, Z is ═O.

R² may be substituted or unsubstituted C₁-C₂₀ alkyl, substituted orunsubstituted 2 to 20 membered heteroalkyl, substituted or unsubstitutedC₃-C₈ cycloalkyl, substituted or unsubstituted 3 to 8 memberedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R² may also be substituted or unsubstitutedC₁-C₁₀ alkyl, substituted or unsubstituted 2 to 10 membered heteroalkyl,substituted or unsubstituted C₃-C₇ cycloalkyl, substituted orunsubstituted 3 to 7 membered heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

In some embodiments, R² is (1) unsubstituted C₁-C₁₀ alkyl; (2)unsubstituted 2 to 10 membered heteroalkyl; (3) unsubstituted C₃-C₇cycloalkyl; (4) unsubstituted 3 to 7 membered heterocycloalkyl; (5)unsubstituted aryl; (6) unsubstituted heteroaryl; (7) substituted C₁-C₁₀alkyl; (8) substituted 2 to 10 membered heteroalkyl; (9) substitutedC₃-C₇ cycloalkyl; (10) substituted 3 to 7 membered heterocycloalkyl;(11) substituted aryl; or (12) substituted heteroaryl. In a relatedembodiment, (7), (8), (9), or (10) (i.e. alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl) is substituted with an oxo, —OH, —CF₃, —COOH, halogen,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl, -L²²-C(O)R³, -L²²-OR⁴,-L²²-NR⁴R⁵, OR⁴, or -L²²-S(O)_(m)R⁶. In another related embodiment whereR² is (11) or (12) (i.e. aryl or heteroaryl), (11) or (12) issubstituted with an —OH, —CF₃, —COOH, halogen, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, R²²-substituted or unsubstitutedheteroaryl, -L²²-C(O)R³, -L²²-OR⁴, -L²²-OR⁴, -L²²-NR⁴R⁵, or-L²²-S(O)_(m)R⁶.

In some embodiments, where (11) or (12) is substituted with aR²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl, thenthe heterocycloalkyl is dioxolanyl, dioxanyl, trioxanyl,tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl,tetrahydrothiopyranyl, pyrrolidinyl, morpholino, piperidinyl, orpiperazinyl. In other embodiments, where (11) or (12) is substitutedwith a R²²-substituted or unsubstituted heteroaryl, then the heteroarylis pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl,pyrrolyl, pyridyl, pyrazyl, pyrimidyl, pyridazinyl, thiazolyl,isothioazolyl, triazolyl, or thienyl, triazinyl, or thiadiazolyl.

R³ is hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl,R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, R²²-substituted or =substituted heteroaryl, —OR³¹,or —NR³²R³³. R³¹, R³², and R³³ are independently hydrogen,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl.

R⁴ and R⁵ are independently hydrogen, —CF₃, R²¹-substituted orunsubstituted alkyl, R²¹-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, R²²-substituted or unsubstitutedheteroaryl, or —C(O)R⁴. R⁴¹ is hydrogen, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl,

R⁶ is hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl,R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or =substituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, or R²²-substituted or unsubstituted heteroaryl.

L²² is a bond, unsubstituted C₁-C₁₀ alkylene or unsubstitutedheteroalkylene. The symbol m represent the integers 0, 1, or 2.

R²¹ is oxo, —OH, —COOH, —CF₃, amino, halogen, R²³-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²³-substituted orunsubstituted C₃-C₇ cycloalkyl, R²³-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²⁴-substituted or unsubstituted aryl, orR²⁴-substituted or unsubstituted heteroaryl. R²² is —OH, —COOH, amino,halogen, —CF₃, R²³-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R²³-substituted or unsubstituted C₃-C₇ cycloalkyl,R²³-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²⁴-substituted or unsubstituted aryl, or R²⁴-substituted orunsubstituted heteroaryl.

In some embodiments, if R²¹ or R²² is substituted or unsubstitutedheterocycloalkyl, the heterocycloalkyl is hydantoinyl, dioxolanyl,dioxanyl, trioxanyl, tetrahydrothienyl, tetrahydrofuranyl,tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl,pyrrolidinyl, morpholino, piperidinyl, or piperazinyl.

In other embodiments, if R²¹ or R²² is substituted or unsubstitutedheteroaryl, then the heteroaryl is pyrazolyl, furanyl, imidazolyl,isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrazyl,pyrimidyl, pyridazinyl, thiazolyl, isothioazolyl, triazolyl, thienyl,triazinyl, or thiadiazolyl.

R²³ is oxo, —OH, —COOH, amino, halogen, —CF₃, unsubstituted C₁-C₁₀alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl. R²⁴ is —OH, —COOH,amino, halogen, —CF₃, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to7 membered heterocycloalkyl, unsubstituted aryl, unsubstitutedheteroaryl.

In some embodiments, R² is substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In other embodiments, R² issubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted alkyl, or substituted orunsubstituted heteroalkyl.

Where R² is substituted (e.g. substituted aryl or heteroaryl), thesubstituent (also referred to herein as the R² substituent) may be a(n):(1) unsubstituted C₁-C₁₀ alkyl; (2) unsubstituted 2 to 10 memberedheteroalkyl; (3) unsubstituted C₃-C₇ cycloalkyl; (4) unsubstituted 3 to7 membered heterocycloalkyl; (5) unsubstituted aryl; (6) unsubstitutedheteroaryl; (7) halogen; (8) —OH; (9) amino; (10) —CF₃; (11) 3 to 7membered heterocycloalkyl substituted with unsubstituted C₁-C₁₀ alkyl;or (12) C₁-C₁₀ alkyl substituted with an unsubstituted aryl. In somerelated embodiments, R² is a substituted aryl or substituted heteroaryl.

In another embodiment, the R² substituent is a: (1) halogen; (2)-L²²-C(O)R³; (3) -L²²-OR⁴; (4) -L²²-NR⁴R⁵; or (5) -L²²-S(O)_(m)R⁶. R³may be hydrogen, unsubstituted C₁-C₁₀ alkyl, —OR³¹, or —NR³²R³³. R³¹,R³², and R³³ may independently be hydrogen, unsubstituted C₁-C₁₀ alkyl,or unsubstituted C₃-C₇ cycloalkyl. L²² may be unsubstituted C₁-C₁₀alkylene. R⁴ may be hydrogen, —CF₃, —CHF₂, unsubstituted C₁-C₁₀ alkyl,unsubstituted C₃-C₇ cycloalkyl, unsubstituted C₁-C₁₀ cycloalkylalkyl, or—C(O)R⁴¹. R⁴¹ may be hydrogen, or unsubstituted C₁-C₁₀ alkyl. R⁴ and R⁵may independently be hydrogen, unsubstituted C₁-C₁₀ alkyl, or —C(O)R⁴¹.R⁴¹ may independently be hydrogen, or unsubstituted C₁-C₁₀ alkyl, or R⁷may be hydrogen or unsubstituted C₁-C₁₀ alkyl.

In some embodiments, where R² is substituted or unsubstituted aryl, thenthe aryl is phenyl, benz[cd]indol-2(1H)-one-yl, oxindolyl,indazolinonyl, benzoimidazolyl, indolyl, benzodioxanyl, coumarinyl,chromonyl, benzopyrazyl, naphthyl, quinolyl, or isoquinolyl.

In other embodiments, where R² is substituted or unsubstitutedheteroaryl, then the heteroaryl is pyrazolyl, furanyl, imidazolyl,isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl,pyrazyl, pyridazinyl, hyadantoin, thiazolyl, isothioazolyl, triazolyl,thienyl, triazinyl, thiadiazolyl.

In certain embodiments, where R² is substituted or unsubstitutedheterocycloalkyl, then the heterocycloalkyl is hydantoinyl, dioxolanyl,dioxanyl, trioxanyl, tetrahydrothienyl, tetrahydrofuranyl,tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl,pyrrolidinyl, morpholino, piperidinyl, or piperazinyl.

R¹ may be hydrogen, amino, substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted 2 to 10 membered heteroalkyl, substitutedor unsubstituted C₃-C₇ cycloalkyl, substituted or unsubstituted 3 to 7membered heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In an exemplary embodiment, R¹ is: (1) —CF₃; (2) unsubstituted C₁-C₁₀alkyl; (3) unsubstituted 2 to 10 membered heteroalkyl; (4)unsubstituted. C₃-C₇ cycloalkyl; (5) unsubstituted 3 to 7 memberedheterocycloalkyl; (6) unsubstituted aryl; (7) unsubstituted heteroaryl;(8) substituted C₁-C₁₀ alkyl; (9) substituted 2 to 10 memberedheteroalkyl; (10) substituted C₃-C₇ cycloalkyl; (11) substituted 3 to 7membered heterocycloalkyl; (12) substituted aryl; or (13) substitutedheteroaryl. In a related embodiment, (8), (9), (10) or (11) issubstituted with an oxo, —OH, —CF₃, —COOH, halogen, R¹⁵-substituted orunsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹⁵-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹⁶-substituted or unsubstituted aryl, R¹⁶-substituted or unsubstitutedheteroaryl, -L¹¹-C(O)R¹⁰⁰, -L¹¹-OR¹⁰⁴, -L¹¹-NR¹⁰⁴R¹⁰⁵, or-L¹¹-S(O)_(q)—R¹⁰⁷. In another related embodiment, (12) or (13) issubstituted with an —OH, —CF₃, —COOH, halogen, R¹⁵-substituted orunsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹⁵-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹⁶-substituted or unsubstituted aryl, R¹⁶-substituted or unsubstitutedheteroaryl, -L¹¹-C(O)R¹⁰⁰, -L¹¹-OR¹⁰⁴, -L¹¹-NR¹⁰⁴R¹⁰⁵, or-L¹¹-S(O)_(q)—R¹⁰⁷.

R¹⁰⁰ is hydrogen, R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl,R¹⁵-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹⁵-substituted or =substituted C₃-C₇ cycloalkyl, R¹⁵-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹⁶-substituted orunsubstituted aryl, R¹⁶-substituted or unsubstituted heteroaryl, —OR¹⁰¹,or —NR¹⁰²R¹⁰³. R¹⁰¹, R¹⁰², and R¹⁰³ are independently hydrogen,R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹⁵-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹⁵-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, orR¹⁶-substituted or unsubstituted heteroaryl.

R¹⁰⁴ and R¹⁰⁵ are independently hydrogen, —CF₃, R¹⁵-substituted orunsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹⁵-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹⁶-substituted or unsubstituted aryl, R¹⁶-substituted or unsubstitutedheteroaryl, or —C(O)R¹⁰⁶. R¹⁰⁶ is independently hydrogen,R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹⁵-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹⁵-substituted or substituted 3 to 7membered heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, orR¹⁶-substituted or unsubstituted heteroaryl.

R¹⁰⁷ is hydrogen, R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl,R¹⁵-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹⁵-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹⁶-substituted orunsubstituted aryl, or R¹⁶-substituted or unsubstituted heteroaryl.

L¹¹ is a bond, unsubstituted C₁-C₁₀ alkylene or unsubstitutedheteroalkylene. The symbol q represents the integers 0, 1, or 2.

R¹⁵ is oxo, —OH, —COOH, —CF₃, halogen, R¹⁷-substituted or unsubstitutedC₁-C₁₀ alkyl, R¹⁷-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R¹⁷-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹⁷-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹⁸-substituted or unsubstituted aryl, or R¹⁸-substituted orunsubstituted heteroaryl. R¹⁶ is —OH, —COOH, —CF₃, halogen,R¹⁷-substituted or unsubstituted C₁-C₁₀ alkyl, R¹⁷-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹⁷-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹⁷-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹⁸-substituted or unsubstituted aryl, orR¹⁸-substituted or unsubstituted heteroaryl.

R¹⁷ is oxo, —OH, —COOH, —CF₃, halogen, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl. R¹⁸ is —OH, —COOH,—CF₃, halogen, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to7 membered heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl.

In some embodiments, R¹⁵ is oxo, —OH, —COOH, —CF₃, halogen,unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl,unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to 7 memberedheterocycloalkyl, unsubstituted aryl, or =substituted heteroaryl. Inother embodiments, R¹⁶ is —OH, —COOH, —CF₃, halogen, unsubstitutedC₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstitutedC₃-C₇ cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.

In certain embodiments, where R¹⁵ or R¹⁶ is substituted or unsubstitutedheterocycloalkyl, the heterocycloalkyl is hydantoinyl, dioxolanyl,dioxanyl, trioxanyl, tetrahydrothienyl, tetrahydrofuranyl,tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl,pyrrolidinyl, morpholino, piperidinyl, or piperazinyl.

In other embodiments, where R¹⁵ or R¹⁶ is substituted or unsubstitutedheteroaryl, the heteroaryl is pyrazolyl, furanyl, imidazolyl,isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrazyl,pyrimidyl, pyridazinyl, thiazolyl, isothioazolyl, triazolyl, or thienyl,triazinyl, or thiadiazolyl.

R¹ may be: (1) unsubstituted C₁-C₁₀ alkyl; (2) unsubstituted 2 to 10membered heteroalkyl; (3) unsubstituted C₃-C₇ cycloalkyl; (4)unsubstituted 3 to 7 membered heterocycloalkyl; (5) substituted C₁-C₁₀alkyl; (6) substituted 2 to 10 membered heteroalkyl; (7) substitutedC₃-C₇ cycloalkyl; (8) substituted 3 to 7 membered heterocycloalkyl; (9)substituted phenyl; or (10) substituted heteroaryl. In some relatedembodiments, (5), (6), (7), or (8) is substituted with an oxo, —OH,—CF₃, —COOH, halogen, R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl, orR¹⁵-substituted or unsubstituted 2 to 10 membered heteroalkyl. In otherrelated embodiments, (9) or (10) is substituted with an —OH, —CF₃,—COOH, halogen, R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl, orR¹⁵-substituted or unsubstituted 2 to 10 membered heteroalkyl.

R¹⁵ may be oxo, —OH, —COOH, —CF₃, halogen, unsubstituted C₁-C₁₀ alkyl,or unsubstituted 2 to 10 membered heteroalkyl.

In an exemplary embodiment, R¹ is: (1) unsubstituted C₁-C₁₀ alkyl; (2)unsubstituted 2 to 10 membered heteroalkyl; (3) unsubstituted C₃-C₇cycloalkyl; (4) unsubstituted 3 to 7 membered heterocycloalkyl; (5)C₁-C₁₀ alkyl substituted with an oxo, —OH, —COOH, —CF₃, halogen,unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl,unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to 7 memberedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; (6) 2to 10 membered heteroalkyl substituted with an oxo, —OH, —CF₃, —COOH,halogen, unsubstituted C₁-C₁₀ alkyl, 2 to 10 membered heteroalkyl,unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to 7 memberedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; (7)C₃-C₇ cycloalkyl substituted with —OH, —CF₃, —COOH, halogen,unsubstituted C₁-C₁₀ alkyl, or unsubstituted 2 to 10 memberedheteroalkyl; (8) 3 to 7 membered heterocycloalkyl substituted with —OH,—CF₃, —COOH, halogen, unsubstituted C₁-C₁₀ alkyl, or unsubstituted 2 to10 membered heteroalkyl; (9) phenyl substituted with —OH, —CF₃, —COOH,halogen, unsubstituted C₁-C₁₀ alkyl, or unsubstituted 2 to 10 memberedheteroalkyl; or (10) heteroaryl substituted with —OH, —CF₃, —COOH,halogen, unsubstituted C₁-C₁₀ alkyl, or unsubstituted 2 to 10 memberedheteroalkyl.

In another exemplary embodiment, where R¹ is a substituted orunsubstituted heteroaryl, the heteroaryl is pyrazolyl, furanyl,imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl,pyrimidyl, pyrazyl, pyridazinyl, thiazolyl, isothioazolyl, triazolyl,thienyl, triazinyl, or thiadiazolyl.

In some embodiments, where R¹ is a substituted or unsubstitutedheterocycloalkyl, then the heterocycloalkyl is hydantoinyl, dioxolanyl,dioxanyl, trioxanyl, tetrahydrothienyl, tetrahydrofuranyl,tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl,pyrrolidinyl, morpholino, piperidinyl, or piperazinyl.

In some embodiments, where R¹ is substituted, the substituent (alsoreferred to herein as an R¹ substituent) is a: (1) halogen; (2)-L¹¹-C(O)R¹⁰⁰; (3) -L¹¹-OR¹⁰⁴; (4)-L¹¹-NR¹⁰⁴R¹⁰⁵; or (5)-L¹¹-S(O)_(q)R¹⁰⁷. In a related embodiment, R¹⁵ is oxo, —OH, —COOH,—CF₃, halogen, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to7 membered heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl, and R¹⁶ is —OH, —COOH, —CF₃, halogen, unsubstituted C₁-C₁₀alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.

II. Exemplary Syntheses

The compounds of the invention are synthesized by an appropriatecombination of generally well known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art, including thetechniques disclosed in Elnagdi, et al., J. Heterocyclic Chem., 16:61-64 (1979), Pawar, et al., Indian J. Chem., 28B: 866-867 (1989),Chande, et al., Indian J. Chem., 35B: 373-376 (1996), and in thefollowing patents DE2429195 (1974), U.S. Pat. No. 6,566,363 (2003),which are incorporated in reference in their entirety for all purposes.The discussion below is offered to illustrate certain of the diversemethods available for use in assembling the compounds of the invention.However, the discussion is not intended to define the scope of reactionsor reaction sequences that are useful in preparing the compounds of thepresent invention. The compounds of this invention may be made by theprocedures and techniques disclosed in the Examples section below, aswell as by known organic synthesis techniques.

In the exemplary syntheses below, the symbols X, R¹⁰, L¹, R¹, R², R¹¹,R¹³, and R¹⁴ are, unless specified otherwise, defined as above in thesection entitled “Bicyclic Pyrazolo Kinase Modulators.”

In step A of general scheme I, synthesis of the required halogenatedintermediates (b), or cyclized intermediates (d) or (m), is accomplishedby reacting a derivative (a), (c) or (k) respectively, with a suitablehalogenating reagent, such as chlorine, bromine or iodine or a suitablehalogen containing reagent such as IC1, N-chlorosuccinimide,N-bromosuccinimide, N-iodosuccinimide, or a tribromide source such asbenzyltrimethylammonium tribromide, in suitable solvents such as aceticacid, DMF, ethereal solvents, or halogenated hydrocarbons, attemperatures ranging from −10° C. to 100° C.

Step B exemplifies cyclization to end products by reacting halogenatedspecies (b) with an acyl isothiocyanate reagent such as benzoylisothiocyanate or furan-2-carbonyl isothiocyanate, and optionally byfurther treatment with a source of sulfur nucleophiles such asLawesson's reagent, in suitable solvents, such as ethereal solvents,DMF, pyridine, or DMSO at temperatures ranging from 20° C. to 180° C.

In step C, synthesis of the required thiourea (c) or urea (e) isperformed by reacting a derivative (a) with thiocarbonyl reagents suchas thiophosgene or thiocarbonyldiimidazole, or carbonyl reagents such asphosgene, triphosgene or carbonyldiimidazole, followed by treatment withammonia or an ammonium source such as ammonium hydroxide, in suitablesolvents such as halogenated hydrocarbons, ethereal solvents, THF, DMF,and water mixtures thereof, at temperatures ranging from −30° C. to 50°C.

Step D exemplifies the synthesis of end products of general formula (I)by reacting intermediate (d), optionally protected at the NH site, withsuitable electrophiles such as acid chlorides, isocyanates,isothiocyanates, sulfonyl chlorides, imidoyl chlorides, imidoate estersor isothioureas, in suitable solvents such as ethereal solvents, DMF,DMSO, at temperatures ranging from 20° C. to 200° C., followed by basichydrolysis with bases such as sodium hydroxide or triethylamine insuitable solvents such as alcohols, ethereal solvents, DMF, and watermixtures thereof, at temperatures ranging from 0° C. to 100° C.

In step E, synthesis of the required nitrated intermediate isaccomplished by reacting derivative (e) with a suitable nitratingreagent such as nitric acid, nitronium salts, ethyl nitrate or nitrogendioxide, in the presence or absence of sulfuric acid or Lewis-acidcatalyst, in suitable solvents such as water, acetic acid, orhalogenated hydrocarbons, at temperatures ranging from −78° C. to 50° C.

Step F shows the exemplary synthesis of the required intermediate (g) byreacting derivative (f) with a suitable reducing agent such as hydrogengas, zinc, iron, tin chloride, or sodium sulfide, in the presence orabsence of a catalyst, such as palladium, or HCl, in suitable solventssuch as water, alcohols, DMF, or ethereal solvents, at temperaturesranging from 20° C. to 200° C.

In step G, synthesis of the required diazonium intermediate (h), ornitroso intermediate (j), is accomplished by reacting derivative (g), or(i) respectively, with a suitable “nitrite” reagent in acidic media,such as sodium nitrite or isoamyl nitrite, in a suitable solvent such aswater or mixture thereof with an organic solvent such as alcohols,ethereal solvents, or DMF, at temperatures ranging from −78° C. to 50°C.

Step H exemplifies synthesis of the required cyclized intermediate (d)by irradiating derivative (h) with a suitable light source such as a 250W mercury lamp, in a suitable solvent such as alcohols, etherealsolvents, halogenated hydrocarbons, or DMF, at temperatures ranging from20° C. to 100° C.

Step I shows the synthesis of the required cyclized intermediate (k) byheating derivative (j) in a suitable solvent such as pyridine, attemperatures ranging from 50° C. to 180° C.

In step J, synthesis of the required intermediate (d) is performed byreacting derivative (m) with ammonia or an ammonium source such asammonium hydroxide, in suitable solvents such as halogenatedhydrocarbons, alcohols, ethereal solvents, or DMF, at temperaturesranging from 20° C. to 180° C.

In addition to general scheme I above, the following exemplary schemesare presented to further illustrate methods of synthesizing the bicyclicpyrazolo kinase modulators of the present invention.

In Scheme 1, methyl ester (a) is reacted with lithiated acetonitrile atlow temperature to give propionitrile (b). Compound (b) is then treatedwith hydrazine to provide aminopyrazole (c).

In Scheme 2, aminopyrazole (a), either commercially available orprepared synthetically as described in Scheme 1, is brominated with asolution of bromine in acetic acid. Brominated pyrazole (b) is thentreated with an acyl isothiocyanate, such as benzoyl isothiocyanate orfuran-2-carbonyl isothiocyanate, to give (1). The cyclization reactionis achieved in either dioxane under thermal conditions, or pyridineunder microwave conditions.

In an alternative method, as shown in Scheme 3, aminopyrazole (a) istreated with thiophosgene in the presence of magnesium oxide and sodiumbicarbonate in dioxane/water mixture, followed by ammonium hydroxide toprovide monosubstituted thiourea (b). Subsequent treatment of (b) withbromine in acetic acid affords cyclized product (c). Compound (c) isreacted with an excess of acyl chloride at reflux temperature, followedby treatment with sodium hydroxide (or other basic hydrolysisconditions, such as Et₃N in THF/H₂O), to afford (la), or (lb) aftertreatment with Lawesson's reagent.

In Scheme 4, pyrazolothiazolamine (a) is protected as a benzamide withbenzoyl chloride to give (b). Subsequent treatment with isocyanate,isothiocyanate, or sulfonyl chloride reagents, followed by basichydrolysis with sodium hydroxide, afford (lc). Similarly, protectedpyrazolothiazolamine (b) is reacted with reagents such as imidoylchlorides (or imidoate ester equivalent) or methylisothioureas to give(ld).

The compounds of the present invention may be synthesized using one ormore protecting groups generally known in the art of chemical synthesis.The term “protecting group” refers to chemical moieties that block someor all reactive moieties of a compound and prevent such moieties fromparticipating in chemical reactions until the protective group isremoved, for example, those moieties listed and described in Greene, etal., Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons(1999). It may be advantageous, where different protecting groups areemployed, that each (different) protective group be removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions allow differential removal of suchprotecting groups. For example, protective groups can be removed byacid, base, and hydrogenolysis. Groups such as trityl, dimethoxytrityl,acetal and t-butyldimethylsilyl are acid labile and may be used toprotect carboxy and hydroxy reactive moieties in the presence of aminogroups protected with Cbz groups, which are removable by hydrogenolysis,and Fmoc groups, which are base labile. Carboxylic acid and hydroxyreactive moieties may be blocked with base labile groups such as,without limitation, methyl, ethyl, and acetyl in the presence of aminesblocked with acid labile groups such as t-butyl carbamate or withcarbamates that are both acid and base stable but hydrolyticallyremovable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be blocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups may be blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with apalladium(0)-catalyzed reaction in the presence of acid labile t-butylcarbamate or base-labile acetate amine protecting groups. Yet anotherform of protecting group is a resin to which a compound or intermediatemay be attached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

Typical blocking or protecting groups include, for example:

III. Methods of Inhibiting Kinases

In another aspect, the present invention provides methods of modulatingprotein kinase activity using the bicyclic pyrazolo kinase modulators ofthe present invention. The term “modulating kinase activity,” as usedherein, means that the activity of the protein kinase is increased ordecreased when contacted with a bicyclic pyrazolo kinase modulator ofthe present invention relative to the activity in the absence of thebicyclic pyrazolo kinase modulator. Therefore, the present inventionprovides a method of modulating protein kinase activity by contactingthe protein kinase with a bicyclic pyrazolo kinase modulator of thepresent invention.

In an exemplary embodiment, the bicyclic pyrazolo kinase modulatorinhibits kinase activity. The term “inhibit,” as used herein inreferenceto kinase activity, means that the kinase activity is decreased whencontacted with a bicyclic pyrazolo kinase modulator relative to theactivity in the absence of the bicyclic pyrazolo kinase modulator.Therefore, the present invention further provides a method of inhibitingprotein kinase activity by contacting the protein kinase with a bicyclicpyrazolo kinase modulator of the present invention.

In certain embodiments, the protein kinase is a protein tyrosine kinase.A protein tyrosine kinase, as used herein, refers to an enzyme thatcatalyzes the phosphorylation of tyrosine residues in proteins with aphosphate donors (e.g. a nucleotide phosphate donor such as ATP).Protein tyrosine kinases include, for example, Abelson tyrosine kinases(“Abl”) (e.g. c-Abl and v-Abl), Ron receptor tyrosine kinases (“RON”),Met receptor tyrosine kinases (“MET”), Fms-like tyrosine kinases (“FLT”)(e.g. FLT3), src-family tyrosine kinases (e.g. lyn, CSK), andp21-activated kinase-4 (“PAK”), FLT3, aurora-A kinases, B-lymphoidtyrosine kinases (“Blk”), cyclin-dependent kinases (“CDK”) (e.g. CDK1and CDK5), src-family related protein tyrosine kinases (e.g. Fynkinase), glycogen synthase kinases (“GSK”) (e.g. GSK3α and GSK3β),lymphocyte protein tyrosine kinases (“Lck”), ribosomal S6 kinases (e.g.Rsk1, Rsk2, and Rsk3), sperm tyrosine kinases (e.g. Yes), and subtypesand homologs thereof exhibiting tyrosine kinase activity. In certainembodiments, the protein tyrosine kinase is Abl, RON, MET, PAK, or FLT3.In other embodiments, the protein tyrosine kinase is a FLT3 or Ablfamily member.

In another embodiment, the kinase is a mutant kinase, such as a mutantAbl kinase or FLT3 kinase. Useful mutant Abl kinases include, forexample, Bcr-Abl and Abl kinases having one of more of the followingmutations: Glu255Lys, Thr315Ile, Tyr293Phe, or Met351Thr. In someembodiments, the mutant Abl kinase has a Y393F mutation or a T315Imutation. In another exemplary embodiment, the mutant Abl kinase has aThr315Ile mutation.

In some embodiments, the kinase is homologous to a known kinase (alsoreferred to herein as a “homologous kinase”). Compounds and compositionsuseful for inhibiting the biological activity of homologous kinases maybe initially screened, for example, in binding assays. Homologousenzymes comprise an amino acid sequence of the same length that is atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90%identical to the amino acid sequence of full length known kinase, or70%, 80%, or 90% homology to the known kinase active domains. Homologymay be determined using, for example, a PSI BLAST search, such as, butnot limited to that described in Altschul, et al., Nuc. Acids Rec.25:3389-3402 (1997). In certain embodiments, at least 50%, or at least70% of the sequence is aligned in this analysis. Other tools forperforming the alignment include, for example, DbClustal and ESPript,which may be used to generate the PostScript version of the alignment.See Thompson et al., Nucleic Acids Research, 28:2919-26, 2000; Gouet, etal., Bioinformatics, 15:305-08 (1999). Homologs may, for example, have aBLAST E-value of 1×10⁻⁶ over at least 100 amino acids (Altschul et al.,Nucleic Acids Res., 25:3389-402 (1997) with FLT3, Abl, or another knownkinase, or any functional domain of FLT3, Abl, or another known kinase.

Homology may also be determined by comparing the active site bindingpocket of the enzyme with the active site binding pockets of a knownkinase. For example, in homologous enzymes, at least 50%, 60%, 70%, 80%,or 90% of the amino acids of the molecule or homolog have amino acidstructural coordinates of a domain comparable in size to the kinasedomain that have a root mean square deviation of the alpha carbon atomsof up to about 1.5 Å, about 1.25 Å, about 1 Å, about 0.75 Å, about 0.5Å, and or about 0.25 Å.

The compounds and compositions of the present invention are useful forinhibiting kinase activity and also for inhibiting other enzymes thatbind ATP. They are thus useful for the treatment of diseases anddisorders that may be alleviated by inhibiting such ATP-binding enzymeactivity. Methods of determining such ATP binding enzymes include thoseknown to those of skill in the art, those discussed herein relating toselecting homologous enzymes, and by the use of the database PROSITE,where enzymes containing signatures, sequence patterns, motifs, orprofiles of protein families or domains may be identified.

The compounds of the present invention, and their derivatives, may alsobe used as kinase-binding agents. As binding agents, such compounds andderivatives may be bound to a stable resin as a tethered substrate foraffinity chromatography applications. The compounds of this invention,and their derivatives, may also be modified (e.g., radiolabelled oraffinity labelled, etc.) in order to utilize them in the investigationof enzyme or polypeptide characterization, structure, and/or function.

In an exemplary embodiment, the bicyclic pyrazolo kinase modulator ofthe present invention is a kinase inhibitor. In some embodiments, thekinase inhibitor has an IC₅₀ of inhibition constant (K_(i)) of less than1 micromolar. In another embodiment, the kinase inhibitor has an IC₅₀ orinhibition constant (K_(i)) of less than 500 micromolar. In anotherembodiment, the kinase inhibitor has an IC₅₀ or K_(i) of less than 10micromolar. In another embodiment, the kinase inhibitor has an IC₅₀ orK_(i) of less than 1 micromolar. In another embodiment, the kinaseinhibitor has an IC₅₀ or K_(i) of less than 500 nanomolar. In anotherembodiment, the kinase inhibitor has an IC₅₀ or K_(i) of less than 10nanomolar. In another embodiment, the kinase inhibitor has an IC₅₀ orK_(i) of less than 1 nanomolar.

IV. Methods of Treatment

In another aspect, the present invention provides methods of treating adisease mediated by kinase activity (kinase-mediated disease ordisorder) in an organism (e.g. mammals, such as humans). By“kinase-mediated” or “kinase-associated” diseases is meant diseases inwhich the disease or symptom can be alleviated by inhibiting kinaseactivity (e.g. where the kinase is involved in signaling, mediation,modulation, or regulation of the disease process). By “diseases” ismeant diseases, or disease symptoms.

Examples of kinase associated diseases include cancer (e.g. leukemia,tumors, and metastases), allergy, asthma, inflammation (e.g.inflammatory airways disease), obstructive airways disease, autoimmunediseases, metabolic diseases, infection (e.g. bacterial, viral, yeast,fungal), CNS diseases, brain tumors, degenerative neural diseases,cardiovascular diseases, and diseases associated with angiogenesis,neovascularization, and vasculogenesis. In an exemplary embodiment, thecompounds are useful for treating cancer, including leukemia, and otherdiseases or disorders involving abnormal cell proliferation,myeloproliferative disorders, hematological disorders, asthma,inflammatory diseases or obesity.

More specific examples of cancers treated with the compounds of thepresent invention include breast cancer, lung cancer, melanoma,colorectal cancer, bladder cancer, ovarian cancer, prostate cancer,renal cancer, squamous cell cancer, glioblastoma, pancreatic cancer,Kaposi's sarcoma, multiple myeloma, and leukemia (e.g. myeloid, chronicmyeloid, acute lymphoblastic, chronic lymphoblastic, Hodgkins, and otherleukemias and hematological cancers).

Other specific examples of diseases or disorders for which treatment bythe compounds or compositions of the invention are useful for treatmentor prevention include, but are not limited to transplant rejection (forexample, kidney, liver, heart, lung, islet cells, pancreas, bone marrow,cornea, small bowel, skin allografts or xenografts and othertransplants), graft vs. host disease, osteoarthritis, rheumatoidarthritis, multiple sclerosis, diabetes, diabetic retinopathy,inflammatory bowel disease (for example, Crohn's disease, ulcerativecolitis, and other bowel diseases), renal disease, cachexia, septicshock, lupus, myasthenia gravis, psoriasis, dermatitis, eczema,seborrhea, Alzheimer's disease, Parkinson's disease, stem cellprotection during chemotherapy, ex vivo selection or ex vivo purging forautologous or allogeneic bone marrow transplantation, ocular disease,retinopathies (for example, macular degeneration, diabetic retinopathy,and other retinopathies), corneal disease, glaucoma, infections (forexample bacterial, viral, or fungal), heart disease, including, but notlimited to, restenosis.

V. Assays

The compounds of the present invention may be easily assayed todetermine their ability to modulate protein kinases, bind proteinkinases, and/or prevent cell growth or proliferation. Some examples ofuseful assays are presented below.

A. Kinase Inhibition and Binding Assays

Inhibition of various kinases is measured by methods known to those ofordinary skill in the art, such as the various methods presented herein,and those discussed in the Upstate KinaseProfiler Assay Protocols June2003 publication.

For example, where in vitro assays are performed, the kinase istypically diluted to the appropriate concentration to form a kinasesolution. A kinase substrate and phosphate donor, such as ATP, is addedto the kinase solution. The kinase is allowed to transfer a phosphate tothe kinase substrate to form a phosphorylated substrate. The formationof a phosphorylated substrate may be detected directly by anyappropriate means, such as radioactivity (e.g. [γ-³²P-ATP]), or the useof detectable secondary antibodies (e.g. ELISA). Alternatively, theformation of a phosphorylated substrate may be detected using anyappropriate technique, such as the detection of ATP concentration (e.g.Kinase-Glo® assay system (Promega)). Kinase inhibitors are identified bydetecting the formation of a phosphorylated substrate in the presenceand absence of a test compound (see Examples section below).

The ability of the compound to inhibit a kinase in a cell may also beassayed using methods well known in the art. For example, cellscontaining a kinase may be contacted with an activating agent (such as agrowth factor) that activates the kinase. The amount of intracellularphosphorylated substrate formed in the absence and the presence of thetest compound may be determined by lysing the cells and detecting thepresence phosphorylated substrate by any appropriate method (e.g.ELISA). Where the amount of phosphorylated substrate produced in thepresence of the test compound is decreased relative to the amountproduced in the absence of the test compound, kinase inhibition isindicated. More detailed cellular kinase assays are discussed in theExamples section below.

To measure the binding of a compound to a kinase, any method known tothose of ordinary skill in the art may be used. For example, a test kitmanufactured by Discoverx (Fremont, Calif.), ED-Staurosporine NSIP™Enzyme Binding Assay Kit (see U.S. Pat. No. 5,643,734) may be used.Kinase activity may also be assayed as in U.S. Pat. No. 6,589,950,issued Jul. 8, 2003.

Suitable kinase inhibitors may be selected from the compounds of theinvention through protein crystallographic screening, as disclosed in,for example Antonysamy, et al., PCT Publication No. WO03087816A1, whichis incorporate herein by reference in its entirety for all purposes.

The compounds of the present invention may be computationally screenedto assay and visualize their ability to bind to and/or inhibit variouskinases. The structure may be computationally screened with a pluralityof compounds of the present invention to determine their ability to bindto a kinase at various sites. Such compounds can be used as targets orleads in medicinal chemistry efforts to identify, for example,inhibitors of potential therapeutic importance (Travis, Science,262:1374, 1993). The three dimensional structures of such compounds maybe superimposed on a three dimensional representation of kinases or anactive site or binding pocket thereof to assess whether the compoundfits spatially into the representation and hence the protein. In thisscreening, the quality of fit of such entities or compounds to thebinding pocket may be judged either by shape complementarity or byestimated interaction energy (Meng, et al., J. Comp. Chem. 13:505-24,1992).

The screening of compounds of the present invention that bind to and/ormodulate kinases (e.g. inhibit or activate kinases) according to thisinvention generally involves consideration of two factors. First, thecompound must be capable of physically and structurally associating,either covalently or non-covalently with kinases. For example, covalentinteractions may be important for designing irreversible or suicideinhibitors of a protein. Non-covalent molecular interactions importantin the association of kinases with the compound include hydrogenbonding, ionic interactions, van der Waals, and hydrophobicinteractions. Second, the compound must be able to assume a conformationand orientation in relation to the binding pocket, that allows it toassociate with kinases. Although certain portions of the compound willnot directly participate in this association with kinases, thoseportions may still influence the overall conformation of the moleculeand may have a significant impact on potency. Conformationalrequirements include the overall three-dimensional structure andorientation of the chemical group or compound in relation to all or aportion of the binding pocket, or the spacing between functional groupsof a compound comprising several chemical groups that directly interactwith kinases.

Docking programs described herein, such as, for example, DOCK, or GOLD,are used to identify compounds that bind to the active site and/orbinding pocket. Compounds may be screened against more than one bindingpocket of the protein structure, or more than one set of coordinates forthe same protein, taking into account different molecular dynamicconformations of the protein. Consensus scoring may then be used toidentify the compounds that are the best fit for the protein (Charifson,P. S. et al., J. Med. Chem. 42: 5100-9 (1999)). Data obtained from morethan one protein molecule structure may also be scored according to themethods described in Klingler et al., U.S. Utility Application, filedMay 3, 2002, entitled “Computer Systems and Methods for VirtualScreening of Compounds.” Compounds having the best fit are then obtainedfrom the producer of the chemical library, or synthesized, and used inbinding assays and bioassays.

Computer modeling techniques may be used to assess the potentialmodulating or binding effect of a chemical compound on kinases. Ifcomputer modeling indicates a strong interaction, the molecule may thenbe synthesized and tested for its ability to bind to kinases and affect(by inhibiting or activating) its activity.

Modulating or other binding compounds of kinases may be computationallyevaluated by means of a series of steps in which chemical groups orfragments are screened and selected for their ability to associate withthe individual binding pockets or other areas of kinases. This processmay begin by visual inspection of, for example, the active site on thecomputer screen based on the kinases coordinates. Selected fragments orchemical groups may then be positioned in a variety of orientations, ordocked, within an individual binding pocket of kinases (Blaney, J. M.and Dixon, J. S., Perspectives in Drug Discovery and Design, 1:301,1993). Manual docking may be accomplished using software such as InsightII (Accelrys, San Diego, Calif.) MOE (Chemical Computing Group, Inc.,Montreal, Quebec, Canada); and SYBYL (Tripos, Inc., St. Louis, Mo.,1992), followed by energy minimization and/or molecular dynamics withstandard molecular mechanics force fields, such as CHARMM (Brooks, etal., J. Comp. Chem. 4:187-217, 1983), AMBER (Weiner, et al., J. Am.Chem. Soc. 106: 765-84, 1984) and C² MMFF (Merck Molecular Force Field;Accelrys, San Diego, Calif.). More automated docking may be accomplishedby using programs such as DOCK (Kuntz et al., J. Mol. Biol., 161:269-88,1982; DOCK is available from University of California, San Francisco,Calif.); AUTODOCK (Goodsell & Olsen, Proteins: Structure, Function, andGenetics 8:195-202, 1990; AUTODOCK is available from Scripps ResearchInstitute, La Jolla, Calif.); GOLD (Cambridge Crystallographic DataCentre (CCDC); Jones et al., J. Mol. Biol. 245:43-53, 1995); and FLEXX(Tripos, St. Louis, Mo.; Rarey, M., et al., J. Mol. Biol. 261:470-89,1996). Other appropriate programs are described in, for example,Halperin, et al.

During selection of compounds by the above methods, the efficiency withwhich that compound may bind to kinases may be tested and optimized bycomputational evaluation. For example, a compound that has been designedor selected to function as a kinases inhibitor may occupy a volume notoverlapping the volume occupied by the active site residues when thenative substrate is bound, however, those of ordinary skill in the artwill recognize that there is some flexibility, allowing forrearrangement of the main chains and the side chains. In addition, oneof ordinary skill may design compounds that could exploit proteinrearrangement upon binding, such as, for example, resulting in aninduced fit. An effective kinase inhibitor may demonstrate a relativelysmall difference in energy between its bound and free states (i.e., itmust have a small deformation energy of binding and/or lowconformational strain upon binding). Thus, the most efficient kinaseinhibitors should, for example, be designed with a deformation energy ofbinding of not greater than 10 kcal/mol, not greater than 7 kcal/mol,not greater than 5 kcal/mol, or not greater than 2 kcal/mol. Kinaseinhibitors may interact with the protein in more than one conformationthat is similar in overall binding energy. In those cases, thedeformation energy of binding is taken to be the difference between theenergy of the free compound and the average energy of the conformationsobserved when the inhibitor binds to the enzyme.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interaction. Examples of programsdesigned for such uses include: Gaussian 94, revision C (Frisch,Gaussian, Inc., Pittsburgh, Pa. ©1995); AMBER, version 7. (Kollman,University of California at San Francisco, ©2002); QUANTA/CHARMM(Accelrys, Inc., San Diego, Calif., ©1995); Insight II/Discover(Accelrys, Inc., San Diego, Calif., ©1995); DelPhi (Accelrys, Inc., SanDiego, Calif., ©1995); and AMSOL (University of Minnesota) (QuantumChemistry Program Exchange, Indiana University). These programs may beimplemented, for instance, using a computer workstation, as are wellknown in the art, for example, a LINUX, SGI or Sun workstation. Otherhardware systems and software packages will be known to those skilled inthe art.

Those of ordinary skill in the art may express kinase protein usingmethods known in the art, and the methods disclosed herein. The nativeand mutated kinase polypeptides described herein may be chemicallysynthesized in whole or part using techniques that are well known in theart (see, e.g., Creighton, Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co., NY, 1983).

Gene expression systems may be used for the synthesis of native andmutated polypeptides. Expression vectors containing the native ormutated polypeptide coding sequence and appropriatetranscriptional/translational control signals, that are known to thoseskilled in the art may be constructed. These methods include in vitrorecombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, NY, 2001, and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and WileyInterscience, NY, 1989.

Host-expression vector systems may be used to express kinase. Theseinclude, but are not limited to, microorganisms such as bacteriatransformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing the coding sequence; yeast transformedwith recombinant yeast expression vectors containing the codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing the coding sequence; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the coding sequence; or animal cell systems. Theprotein may also be expressed in human gene therapy systems, including,for example, expressing the protein to augment the amount of the proteinin an individual, or to express an engineered therapeutic protein. Theexpression elements of these systems vary in their strength andspecificities.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector may contain: an origin of replication forautonomous replication in host cells, one or more selectable markers, alimited number of useful restriction enzyme sites, a potential for highcopy number, and active promoters. A promoter is defined as a DNAsequence that directs RNA polymerase to bind to DNA and initiate RNAsynthesis. A strong promoter is one that causes mRNAs to be initiated athigh frequency.

The expression vector may also comprise various elements that affecttranscription and translation, including, for example, constitutive andinducible promoters. These elements are often host and/or vectordependent. For example, when cloning in bacterial systems, induciblepromoters such as the T7 promoter, pL of bacteriophage λ, plac, ptrp,ptac (ptrp-lac hybrid promoter) and the like may be used; when cloningin insect cell systems, promoters such as the baculovirus polyhedrinpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll a/b binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, mammalian promoters (e.g.,metallothionein promoter) or mammalian viral promoters, (e.g.,adenovirus late promoter; vaccinia virus 7.5K promoter; SV40 promoter;bovine papilloma virus promoter; and Epstein-Barr virus promoter) may beused.

Various methods may be used to introduce the vector into host cells, forexample, transformation, transfection, infection, protoplast fusion, andelectroporation. The expression vector-containing cells are clonallypropagated and individually analyzed to determine whether they producethe appropriate polypeptides. Various selection methods, including, forexample, antibiotic resistance, may be used to identify host cells thathave been transformed. Identification of polypeptide expressing hostcell clones may be done by several means, including but not limited toimmunological reactivity with anti-kinase antibodies, and the presenceof host cell-associated activity.

Expression of cDNA may also be performed using in vitro producedsynthetic mRNA. Synthetic mRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell-basedsystems, including, but not limited, to microinjection into frogoocytes.

To determine the cDNA sequence(s) that yields optimal levels of activityand/or protein, modified cDNA molecules are constructed. A non-limitingexample of a modified cDNA is where the codon usage in the cDNA has beenoptimized for the host cell in which the cDNA will be expressed. Hostcells are transformed with the cDNA molecules and the levels of kinaseRNA and/or protein are measured.

Levels of kinase protein in host cells are quantitated by a variety ofmethods such as immunoaffinity and/or ligand affinity techniques,kinase-specific affinity beads or specific antibodies are used toisolate 35S-methionine labeled or unlabeled protein. Labeled orunlabeled protein is analyzed by SDS-PAGE. Unlabeled protein is detectedby Western blotting, ELISA or RIA employing specific antibodies.

Following expression of kinase in a recombinant host cell, polypeptidesmay be recovered to provide the protein in active form. Severalpurification procedures are available and suitable for use. Recombinantkinase kinase may be purified from cell lysates or from conditionedculture media, by various combinations of, or individual application of,fractionation, or chromatography steps that are known in the art.

In addition, recombinant kinase can be separated from other cellularproteins by use of an immuno-affinity column made with monoclonal orpolyclonal antibodies specific for full length nascent protein orpolypeptide fragments thereof. Other affinity based purificationtechniques known in the art may also be used.

Alternatively, the polypeptides may be recovered from a host cell in anunfolded, inactive form, e.g., from inclusion bodies of bacteria.Proteins recovered in this form may be solubilized using a denaturant,e.g., guanidinium hydrochloride, and then refolded into an active formusing methods known to those skilled in the art, such as dialysis.

B. Cell Growth Assays

A variety of cell growth assays are known in the art and are useful inidentifying bicyclic pyrazolo compounds (i.e. “test compounds”) capableof inhibiting (e.g. reducing) cell growth and/or proliferation.

For example, a variety of cells are known to require specific kinasesfor growth and/or proliferation. The ability of such a cell to grow inthe presence of a test compound may be assessed and compared to thegrowth in the absence of the test compound thereby identifying theanti-proliferative properties of the test compound. One common method ofthis type is to measure the degree of incorporation of label, such astritiated thymidine, into the DNA of dividing cells. Alternatively,inhibition of cell proliferation may be assayed by determining the totalmetabolic activity of cells with a surrogate marker that correlates withcell number. Cells may be treated with a metabolic indicator in thepresence and absence of the test compound. Viable cells metabolize themetabolic indicator thereby forming a detectable metabolic product.Where detectable metabolic product levels are decreased in the presenceof the test compound relative to the absence of the test compound,inhibition of cell growth and/or proliferation is indicated. Exemplarymetabolic indicators include, for example tetrazolium salts andAlamorBlue® (see Examples section below).

An assay for kinases that stimulate cell migration is the scratch assay.This assay is used to evaluate inhibitors of kinases by mimicking eventssuch as wound healing. In one variant of this assay used to test METinhibitors, a confluent monolayer of cells is allowed to form on a cellplate. After formation of the monolayer, a linear wound on the monolayeris generated by mechanically scraping the monolayer thereby forming acell-free channel. A growth factor required by the kinase for cellgrowth is added in the presence or absence of the test compound. Theclosure of the channel in the presence of the test compound indicates afailure of the test compound to inhibit the kinase thereby allowing cellmigration and growth to close the channel. Conversely, the presence ofthe channel after adding the test compound indicates that test compoundinhibited the kinase thereby preventing cell growth. The selection ofthe appropriate cells, growth conditions, and growth factors are wellwithin the abilities of one skilled in the art (see Examples sectionbelow).

VI. Pharmaceutical Compositions and Administration

In another aspect, the present invention provides a pharmaceuticalcomposition including a bicyclic pyrazolo kinase modulator in admixturewith a pharmaceutically acceptable excipient. One of skill in the artwill recognize that the pharmaceutical compositions include thepharmaceutically acceptable salts of the bicyclic pyrazolo kinasemodulators described above.

In therapeutic and/or diagnostic applications, the compounds of theinvention can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams & Wilkins(2000).

The compounds according to the invention are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, andfrom 5 to 40 mg per day are examples of dosages that may be used. A mostpreferable dosage is 10 to 30 mg per day. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Preferred pharmaceuticallyacceptable salts include, for example, acetate, benzoate, bromide,carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate,mesylate, napsylate, pamoate (embonate), phosphate, salicylate,succinate, sulfate, or tartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Suitable routes may include oral,buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal,transmucosal, nasal or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intra-articullar, intra-sternal, intra-synovial, intra-hepatic,intralesional, intracranial, intraperitoneal, intranasal, or intraocularinjections or other modes of delivery.

For injection, the agents of the invention may be formulated and dilutedin aqueous solutions, such as in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For such transmucosal administration, penetrants appropriate to thebather to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the invention intodosages suitable for systemic administration is within the scope of theinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present invention, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

For nasal or inhalation delivery, the agents of the invention may alsobe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Depending upon the particular condition, or disease state, to be treatedor prevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may be administeredtogether with the inhibitors of this invention. For example,chemotherapeutic agents or other anti-proliferative agents may becombined with the inhibitors of this invention to treat proliferativediseases and cancer. Examples of known chemotherapeutic agents include,but are not limited to, adriamycin, dexamethasone, vincristine,cyclophosphamide, fluorouracil, topotecan, taxol, interferons, andplatinum derivatives.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation, anti-inflammatory agents suchas corticosteroids, TNF blockers, IL-1 RA, azathioprine,cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and anti-viral agents;agents for treating blood disorders such as corticosteroids,anti-leukemic agents, and growth factors; agents for treating diabetessuch as insulin, insulin analogues, alpha glucosidase inhibitors,biguanides, and insulin sensitizers; and agents for treatingimmunodeficiency disorders such as gamma globulin.

These additional agents may be administered separately, as part of amultiple dosage regimen, from the inhibitor-containing composition.Alternatively, these agents may be part of a single dosage form, mixedtogether with the inhibitor in a single composition.

The present invention is not to be limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention. Indeed, various modifications of the invention inaddition to those described herein will become apparent to those havingskill in the art from the foregoing description. Such modifications areintended to fall within the scope of the invention. Moreover, any one ormore features of any embodiment of the invention may be combined withany one or more other features of any other embodiment of the invention,without departing from the scope of the invention. For example, thebicyclic pyrazolo kinase modulators described in the Bicyclic PyrazoloKinase Modulators section are equally applicable to the methods oftreatment and methods of inhibiting kinases described herein. Referencescited throughout this application are examples of the level of skill inthe art and are hereby incorporated by reference herein in theirentirety for all purposes, whether previously specifically incorporatedor not.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. The preparation of embodiments of the presentinvention is described in the following examples. Those of ordinaryskill in the art will understand that the chemical reactions andsynthesis methods provided may be modified to prepare many of the othercompounds of the present invention. Where compounds of the presentinvention have not been exemplified, those of ordinary skill in the artwill recognize that these compounds may be prepared by modifyingsynthesis methods presented herein, and by using synthesis methods knownin the art.

Example 1 Synthesis of Compounds Synthesis of3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-ylamine

To a stirring solution of 5-amino-3-phenylpyrazole (5 g, 31.4 mmol) indioxane (100 mL) was added MgO (1.27 g, 31.4 mmol) and a saturatedaqueous solution of sodium bicarbonate (100 mL). The heterogeneousmixture was stirred for 30 min, then thiophosgene (2.65 mL, 34.6 mmol)was added dropwise, and the reaction mixture was further stirredvigorously for 15 min until completion. Ammonium hydroxide (25 mL) wasthen added dropwise, and the reaction mixture was stirred for another 20min. A 10% aqueous solution of citric acid was added to lower the pH to6, and the mixture was extracted with ethyl acetate (3×). The organiclayer was dried over sodium sulfate, filtered, and adsorbed on silicagel. Purification on silica gel with 10-80% ethyl acetate in hexane aseluent provided 6.22 g (91%) of (5-Phenyl-2H-pyrazol-3-yl)-thiourea as ayellow foam.

The yellow foam (6.2 g, 28.4 mmol) was dissolved in AcOH (125 mL) and a1.5M solution of bromine in AcOH (20.8 mL, 31.2 mmol) was added dropwiseover 30 min under vigorous stirring. The resulting heterogeneous mixturewas stirred at 80° C. for 2.5 h, then cooled to room temperature, andconcentrated in vacuo. The residue was suspended in water and 4N aqueousNaOH was added until pH 7. After extraction with ethyl acetate, theorganic layer was adsorbed on silica gel. Purification on silica gelwith 0-10% MeOH in CH₂Cl₂ as eluent provided 3.26 g of a yellow solidthat was further recrystallized from isopropyl alcohol to yield 2.81 g(45%) of pure 3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-ylamine as off-whitecrystals. ¹H-NMR (d₆-DMSO) δ: 12.8 and 12.9 (2 broad s, 1H,NH+tautomer), 7.60 (broad s, 2H), 7.55 (broad s, 2H, NH₂), 7.48 (broads; 2H), 7.33 (t, 1H); HPLC/MS m/z: 217 [MH]⁺.

Synthesis ofN-(3-thiophen-2-yl-1H-pyrazolo[3,4-d]thiazol-5-yl)-benzamide

To a stirring solution of 5-amino-3-(2-thienyl)pyrazole (500 mg, 3.03mmol) in THF (10 mL) was added N-bromosuccinimide (592 mg, 3.33 mmol) inone portion. The reaction mixture was stirred at room temperature for 3h, then the solvent was evaporated and the residue was taken up in ethylacetate. The organic layer was washed successively with 1M aqueoussodium thiosulfate, twice with a saturated aqueous solution of sodiumbicarbonate, and brine, then it was dried over sodium sulfate, filtered,and adsorbed on silica gel. Purification on silica gel with 0-10% MeOHin CH₂Cl₂ as eluent provided 686 mg (93%) of4-bromo-5-thiophen-2-yl-2H-pyrazol-3-ylamine as a dark foam. ¹H-NMR(d₆-DMSO) δ: 11.9 and 12.4 (2 broad s, 1H, NH+tautomer), 7.4-7.65 (m,2H), 7.05-7.2 (m, 1H), 4.8 and 5.3 (2 broad s, 2H, NH₂+tautomer).

To a stirring solution of 4-bromo-5-thiophen-2-yl-2H-pyrazol-3-ylamine(50 mg, 0.205 mmol) in dioxane (1 mL) was added benzoylisothiocyanate(30 μL, 0.225 mmol) dropwise. The reaction mixture was stirred at 90° C.for 17 h. It was then cooled to room temperature and partitioned betweenethyl acetate and a saturated aqueous solution of sodium bicarbonate.The organic layer was washed twice with a saturated aqueous solution ofsodium bicarbonate, then with brine, and adsorbed on silica gel.Purification on silica gel with 0-80% ethyl acetate in hexane as eluentprovided 54 mg (80%) ofN-(3-thiophen-2-yl-1H-pyrazolo[3,4-d]thiazol-5-yl)-benzamide as a beigesolid. ¹H-NMR (d₆-DMSO) δ: 13.5 and 13.6 (2 broad s, 111, NH+tautomer),12.9 (broad s, 1H, NH), 8.1 (d, 2H), 7.68 and 7.85 (2 dd, 1H,tautomers), 7.66 (t, 1H), 7.58 (t, 2H), 7.34 and 7.53 (2 d, 1H,tautomers), 7.17 and 7.22 (2 t, 1H, tautomers); HPLC/MS m/z: 327 [MH]⁺.

Synthesis ofN-[3-(3-chloro-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-yl]-benzamide

To a stirring solution of 3-(3-chlorophenyl)-3-oxo-propionitrile (2 g,11.1 mmol) in absolute ethanol (20 mL) was added hydrazine hydrate (3.24mL, 67 mmol). The reaction mixture was stirred at 80° C. for 15 h,cooled to room temperature, concentrated in vacuo, and absorbed onsilica gel. Purification on silica gel with 0-10% MeOH in CH₂Cl₂ aseluent provided 1.53 g (70%) of 5-(3-chloro-phenyl)-2H-pyrazol-3-ylamineas a green solid. ¹H NMR (d₆-DMSO) δ: 11.6 and 11.9 (2 broad s, 1H,NH+tautomer), 7.69 (s, 1H), 7.60 (d, 1H), 7.38 (t, 1H), 7.29 (d, 1H),5.77 (broad s, 1H), 4.98 (broad s, 2H, NH₂).

To a stirring solution of 5-(3-chloro-phenyl)-2H-pyrazol-3-ylamine (1.53g, 7.9 mmol) in THF (32 mL) was added N-bromosuccinimide (1.55 g, 8.7mmol). The reaction mixture was stirred for 5 h at room temperature,then absorbed on silica gel. Purification on silica gel with 0-10% MeOHin CH₂Cl₂ as eluent provided 1.73 g (80%) of4-bromo-5-(3-chloro-phenyl)-2H-pyrazol-3-ylamine as a brown solid. ¹HNMR (d₆-DMSO) δ: 12.2 and 12.4 (2 broad s, 1H, NH+tautomer), 7.77 (s,1H), 7.73 (broad s, 1H), 7.48 (broad s, 1H), 7.43 (broad s, 1H), 5.30(broad s, 1H, NH), 4.82 (broad s, 1H, NH); HPLC/MS m/z: 272 [MH]⁺.

To a stirring solution of4-bromo-5-(3-chloro-phenyl)-2H-pyrazol-3-ylamine (50 mg, 0.183 mmol) indioxane (1 mL) was added benzoyl isothiocyanate (27 μL, 0.202 mmol). Thereaction mixture was stirred overnight at 90° C., cooled at roomtemperature, and partitioned between ethyl acetate and sodiumbicarbonate. The organic layer was isolated and absorbed on silica gel.Purification on silica gel with 0-10% MeOH in CH₂Cl₂ as eluent provided10.8 mg (17%) ofN-[3-(3-chloro-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-yl]-benzamide as awhite solid. ¹H NMR (d₆-DMSO) δ: 13.70 and 13.72 (2 broad s, 1H,NH+tautomer), 12.9 (broad s, 1H, NH), 8.12 (d, 2H), 7.81 and 7.85 (2broad s, 1H, tautomers), 7.65-7.74 (m, 2H), 7.54-7.62 (m, 3H), 7.45 (m,1H); HPLC/MS m/z: 355 [MH]⁺.

Synthesis of N-(3-pyridin-2-yl-1H-pyrazolo[3,4-d]thiazol-5-yl)-benzamide

Acetonitrile (0.87 mL) was added dropwise to a mixture of n-butyllithium (2.5 M in hexanes, 6.64 mL) and THF (15 mL) at −78° C. Themixture was left to stir for 1 h. A solution of ethyl picolinate (2.7 g,15.1 mmol) in THF (15 mL) was added dropwise, maintaining thetemperature at −78° C. Stirring was continued at −78° C. for anadditional 2 h. The mixture was allowed to warm up to room temperatureand stirring was continued for an additional 90 min. The reaction wasquenched by addition of water (20 mL). The pH of the solution wasadjusted to 4 with a 1 N aqueous solution of HCl and the aqueoussolution extracted with EtOAc. The combined organic extracts were driedover MgSO₄, filtered, and the solvent removed under reduced pressure.The crude product was dissolved in AcOH (8 mL) and Br₂ (0.22 mL, 4.6mmol) was added dropwise. The mixture was stirred over the course of onehour. Filtration followed by washing with Et₂O afforded 500 mg (10%) of4-bromo-5-pyridin-2-yl-2H-pyrazol-3-ylamine hydrobromide salt as a darksolid. ¹H NMR (d₆-DMSO) δ: 9.12 (s, 1H), 8.82 (m, 1H), 8.76 (m, 1H),8.04 (m, 1H); HPLC/MS m/z: 239 [MH]⁺.

A mixture of 4-bromo-5-pyridin-2-yl-2H-pyrazol-3-ylamine hydrobromidesalt (300 mg, 0.94 mmol), benzoyl isothiocyanate (0.15 mL, 1.12 mmol),pyridine (0.24 mL, 2.97 mmol), 1,4-dioxane (2 mL), and DMSO (2 mL) washeated at 95° C. overnight. The mixture was added to an excess of water,and the resulting precipitate was filtered, and recrystallized from EtOHto afford 190 mg (63%) ofN-(3-pyridin-2-yl-1H-pyrazolo[3,4-d]thiazol-5-yl)-benzamide as anoff-white solid. ¹H NMR (d₆-DMSO) a 8.85 (s, 1H), 8.02 (m, 4H), 7.51 (m,3H), 7.21 (m, 1H); HPLC/MS m/z: 322 [MH]⁺.

Synthesis of N-(3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-isonicotinamide

To a stirring solution of 3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-ylamine(20 mg, 0.092 mmol) and pyridine (52 μL, 0.65 mmol) in THY (0.5 mL) wasadded isonicotinoyl chloride hydrochloride (82 mg, 0.46 mmol). Thereaction mixture was stirred at 70° C. for 22 h, then cooled to roomtemperature and treated with MeOH (1 mL) and a 4N aqueous solution ofNaOH (0.25 mL). The reaction mixture was further stirred at roomtemperature for 4 h, then a 1N aqueous solution of HCl was added toadjust the pH to 7, and the mixture was extracted with 10% MeOH inCH₂Cl₂ (3×). Purification on silica gel with 0-10% MeOH in CH₂Cl₂ aseluent provided 12.5 mg (42%) ofN-(3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-isonicotinamide as a beigesolid. ¹H-NMR (d₆-DMSO) δ: 13.5 and 13.7 (2 broad s, 1H, NH+tautomer),13.2 (broad s, 1H, NH), 8.83 (d, 2H), 7.99 (d, 2H), 7.80 (m, 2H,tautomers), 7.55 (m, 2H, tautomers), 7.40 (m, 1H, tautomers; HPLC/MSm/z: 322 [MH]⁺.

Synthesis of furan-2-carboxylic acid(3-tert-butyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-amide

To a stirring solution of3-tert-Butyl-1H-pyrazolo[3,4-d]thiazol-5-ylamine (30 mg, 0.153 mmol) andpyridine (74 μL, 0.92 mmol), or alternatively PS-DMAP (Argonaut resin, 6equiv.), in THF (0.75 mL) was added furan-2-carbonyl chloride (75 μL,0.76 mmol). The reaction mixture was stirred at 70° C. for 23 h, thencooled to room temperature and treated with THF (1 mL) and PS-trisamine(Argonaut resin, 20 equiv.) for 2 h. The resin was filtered, washed withDMF and the solvent was evaporated. The residue was treated with MeOH (1mL) and a 4N aqueous solution of NaOH (0.25 mL) and the solution wasstirred at room temperature for 2 h. The reaction mixture wasneutralized to pH 7 with a 1N aqueous solution of HCl and extracted withethyl acetate (3×). Purification on silica gel with 0-10% MeOH in CH₂Cl₂as eluent provided 36 mg (82%) of furan-2-carboxylic acid(3-tert-butyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-amide as an off-whitesolid. ¹H-NMR (d₆-DMSO) δ: 12.8 (broad s, 1H, NH), 12.7 (broad s, 1H,NH), 8.03 (d, 1H), 7.69 (broad s, 1H), 6.74 (dd, 1H), 1.36 (s, 9H);HPLC/MS m/z: 291 [MH]⁺.

Synthesis of2-(2-methoxy-ethoxy)-N-(3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-acetamide

To a vial charged with 3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-ylamine (30mg, 0.139 mmol), PS-DMAP (Argonaut resin, 6 equiv.), and a stirring barwas added THF (2 mL) and 2-(2-methoxyethoxy)acetyl chloride (106 μL, ca.5 equiv.). The reaction mixture was stirred at 70° C. for 24 h, thencooled to room temperature and treated with PS-trisamine (Argonautresin, 20 equiv.) for 1.5 h. The resin was filtered, washed with THF andthe solvent was evaporated. The residue was treated with a (5:1:1)mixture of THF/H₂O/Et₃N (2 mL) at 50° C. for 24 h, then the solution wasdirectly adsorbed on silica gel. Purification on silica gel with 0-10%MeOH in CH₂Cl₂ as eluent provided 30 mg (65%) of2-(2-methoxy-ethoxy)-N-(3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-acetamideas a white solid. ¹H-NMR (d₆-DMSO) δ: 13.5 and 13.6 (2 broad s, 1H,NH+tautomer), 12.2 (broad s, 1H, NH), 7.74 (m, 2H, tautomers), 7.54 (m,2H, tautomers), 7.38 (m, 1H, tautomers), 4.28 (s, 2H), 3.68 (t, 2H),3.51 (t, 2H), 3.29 (s, 3H); HPLC/MS m/z: 333 [MH]⁺.

Synthesis of furan-2-carboxylic acid(3-pyridin-2-yl-1H-pyrazolo[3,4-d]thiazol-5-yl)-amide

A mixture of 4-bromo-5-pyridin-2-yl-2H-pyrazol-3-ylamine hydrobromidesalt (193 mg, 0.60 mmol), furan-2-carbonyl isothiocyanate (0.07 mL),pyridine (0.1 mL), and DMSO (2 mL) was placed in a Smith process vial.The reaction was run in a Personal Chemistry SmithCreator microwave at160° C. for five minutes. The reaction mixture was then added to anexcess of water and the resulting precipitate was filtered. Purificationby preparative HPLC afforded 10 mg of furan-2-carboxylic acid(3-pyridin-2-yl-1H-pyrazolo[3,4-d]thiazol-5-yl)-amide as a white solid.¹H-NMR (d₆-DMSO) δ: 8.60 (dd, 1H), 7.92 (m, 2H), 7.80 (d, 1H), 7.46 (d,1H), 7.26 (m, 2H), 6.60 (dd, 1H); HPLC/MS m/z: 312 [MH]⁺.

Synthesis of 2-chloro-4-methyl-thiazole-5-carboxylic acid(3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-amide

To a vial equipped with a teflon cap was added2-bromo-4-methyl-thiazole-5-carboxylic acid (350 mg, 1.576 mmol),thionyl chloride (3 mL), and DMF (0.02 mL). The reaction mixture wasstirred at 85° C. for 3 hours, then the excess of thionyl chloride wasevaporated in vacuo, dry toluene was added, and the solvent wasevaporated in vacuo. Dry toluene (3 mL) was added to generate a solutionof 2-chloro-4-methyl-thiazole-5-carbonyl chloride (ca. 0.52 M) that wasused as such in the next step.

To a vial charged with 3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-ylamine (30mg, 0.139 mmol), PS-DMAP (Argonaut resin, 558 mg, 6 equiv.), and astirring bar was added THF (2 mL) and2-chloro-4-methyl-thiazole-5-carbonyl chloride solution in toluene (0.52M, 1.3 mL, ca. 5 equiv.). The reaction mixture was stirred at 70° C. for18 hours, then cooled to room temperature and treated with PS-trisamine(Argonaut resin, 674 mg, 20 equiv.) for 2 hours. The resin was filtered,washed with THF and the solvent was evaporated. The resulting crudemixture was treated with MeOH (1 mL) and a 4 N aqueous solution of NaOH(0.25 mL). The reaction mixture was further stirred at room temperaturefor 2 hours, then a 1 N aqueous solution of HCl was added to adjust thepH to 7, and the mixture was extracted with EtOAc (3×). Purification onsilica gel with 0-10% MeOH in CH₂Cl₂ as eluent provided 11 mg (21%yield) of 2-chloro-4-methyl-thiazole-5-carboxylic acid(3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-amide as an off-white solid.¹H-NMR (d₆-DMSO) δ: 7.75 (d, 2H), 7.54 (t, 2H), 7.39 (t, 1H), 2.65 (s,3H); HPLC/MS m/z: 376.0 [MH]⁺.

Synthesis ofN-[3-(4-methoxy-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-yl]-acetamide

To a solution of 3-(4-methoxyphenyl)-3-oxo-propionitrile (2.0 g, 11.4mmol) in absolute ethanol (21 mL) was added hydrazine hydrate (3.32 mL,68.3 mmol). The reaction mixture was stirred at 80° C. for 15 hours,then it was concentrated in vacuo and purified on silica gel with 0-10%MeOH in CH₂Cl₂ as eluent to give 2.02 g (97% yield) of5-amino-3-(4-methoxyphenyl)pyrazole as a white solid. ¹H NMR (d₆-DMSO)δ: 11.69 (s, 1H), 7.55 (d, 2H), 6.92 (d, 2H), 5.66 (s, 1H), 4.62 (broads, 2H), 3.75 (s, 3H); HPLC/MS m/z: 190.1

To a solution of 5-amino-3-(4-methoxyphenyl)pyrazole (2.5 g, 13.2 mmol)in THF (50 mL) was added dropwise benzoyl isothiocyanate (1.96 mL, 14.5mmol). The reaction mixture was stirred at room temperature for 45minutes, then 4 N aqueous solution of NaOH (10 mL) was added, and thereaction mixture was further stirred at 55° C. for 1 hour. The reactionmixture was cooled to room temperature, neutralized to pH 8 with 1 Naqueous HCl, and extracted with EtOAc (3×). The combined organic layerswere washed with brine, dried over sodium sulfate, filtered, evaporated,and dried in vacuo to provide 4.28 g of an orange-yellow solid. Thesolid was dissolved in glacial AcOH (300 mL) and a 1.5 M solution ofbromine in AcOH (8.8 mL, 13.2 mmol) was added dropwise under vigorousstirring. The resulting heterogeneous mixture was stirred at roomtemperature for 1 hour then at 80° C. for 1 hour. The reaction wascooled to room temperature and Et₂O (500 mL) was added. The resultingprecipitate was filtered, washed with Et₂O, and dried in vacuo to afford2.93 g (68% yield) of3-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine as a lightyellow solid. ¹H-NMR (d₆-DMSO) δ: 7.57 (d, 2H), 7.09 (d, 2H), 3.80 (s,3H); HPLC/MS m/z: 247.1 [MH]⁺.

To a vial charged with3-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine (100 mg, 0.306mmol), PS-DMAP (Argonaut resin, 1.2 g, 6 equiv.), and a stirring bar wasadded THF (3 mL) and acetyl chloride (110 μL, 1.53 mmol). The reactionmixture was stirred at 70° C. for 1 hour, then cooled to roomtemperature and treated with PS-trisamine (Argonaut resin, 1.1 g, 20equiv.) for 1 hour. The resin was filtered, washed with THF and thesolvent was evaporated. The resulting crude was suspended in MeOH andtreated with hydrazine monohydrate (0.06 mL) for 30 minutes. Theprecipitate was filtered, and the filtrate was directly purified onsilica gel with 0-10% MeOH in CH₂Cl₂ as eluent to provide 12.5 mg (14%yield) ofN-[3-(4-methoxy-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-yl]-acetamide as anoff-white solid. ¹H-NMR (d₆-DMSO) δ: 13.4 (broad s, 1H), 12.3 (broad s,1H), 7.66 (broad s, 2H), 7.09 (broad s, 2H), 3.80 (s, 3H), 2.18 (s,31-1); HPLC/MS m/z: 289.0 [MH]⁺.

Synthesis of2-methoxy-N-(3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-acetamide

To a vial charged with 3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-ylamine (30mg, 0.139 mmol), PS-DMAP (Argonaut resin, 0.56 g, 6 equiv.), and astirring bar was added THF (2 mL) and 2-methoxyacetyl chloride (63 μL,0.694 mmol). The reaction mixture was stirred at 70° C. for 3.5 hours,then cooled to room temperature and treated with PS-trisamine (Argonautresin, 0.51 g, 15 equiv.) at 50° C. for 3 hours. The resin was filtered,washed with THF and the solvent was evaporated. Purification on silicagel with 0-10% MeOH in CH₂Cl₂ as eluent provided 15 mg (37% yield) of2-methoxy-N-(3-phenyl-1H-pyrazolo[3,4-d]thiazol-5-yl)-acetamide as awhite solid. ¹H-NMR (d₆-DMSO) δ: 13.6 (broad s, 1H), 12.3 (broad s, 1H),7.73 (broad s, 2H), 7.53 (broad s, 2H), 7.37 (broad s, 1H), 4.20 (s,2H), 3.37 (s, 3H), 2.18 (s, 3H); HPLC/MS m/z: 289.1 [MH]⁺.

Synthesis of 5-morpholin-4-ylmethyl-furan-2-carboxylic acid[3-(2-bromo-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-yl]-amide

To a solution of ethyl 5-(chloromethyl)-2-furan-carboxylate (0.5 mL,3.25 mmol) and Et₃N (0.9 mL, 6.5 mmol) in dichloromethane under nitrogenwas added morpholine (284 μL, 3.25 mmol) dropwise and a catalytic amountof KI. The reaction mixture was stirred at 45° C. for 24 hours, then itwas concentrated in vacuo. The residue was dissolved in EtOAc and theorganic layer was washed with water (2×) then brine, dried over sodiumsulfate, filtered, concentrated, and dried in vacuo to give 520 mg (67%yield) of 5-morpholin-4-ylmethyl-furan-2-carboxylic acid ethyl ester asa light brown oil. ¹H-NMR (d₆-DMSO) δ: 7.22 (d, 1H), 6.51 (d, 1H), 4.25(q, 2H), 3.54 (m, 6H), 2.37 (broad s, 4H), 1.27 (t, 3H).

To a solution of 5-morpholin-4-ylmethyl-furan-2-carboxylic acid ethylester (510 mg, 2.13 mmol) in MeOH (20 mL) was added Amberlyst A26(OH)(10 g, 21.3 mmol), and the reaction mixture was shaken for 24 hours. Theresin was filtered, washed with MeOH, then taken into 1.25 M HCl in MeOH(50 ml). The resin was filtered, washed with MeOH, and the solution wasevaporated to dryness to give 421 mg (80% yield) of5-morpholin-4-ylmethyl-furan-2-carboxylic acid hydrochloride as a foam.¹H-NMR (d₆-DMSO) δ: 11.54 (broad s, 1H), 7.26 (d, 1H), 6.92 (d, 1H),4.49 (broad s, 2H), 3.93 (broad s, 2H), 3.74 (broad s, 2H), 3.27 (broads, 2H), 3.09 (broad s, 2H).

A suspension of 5-morpholin-4-ylmethyl-furan-2-carboxylic acidhydrochloride in thionyl chloride with 2 drops of DMF was refluxed underN₂ for 3 hours, then cooled to room temperature. Dry CH₂Cl₂ was addedand solvents were evaporated in vacuo. The residue was triturated withdry CH₂Cl₂, and the resulting solid was filtered, washed with dry CH₂Cl₂and dried in vacuo to give 373 mg (83% yield) of5-morpholin-4-ylmethyl-furan-2-carbonyl chloride hydrochloride as awhite solid. ¹H-NMR (d₆-DMSO) δ: 11.54 (broad s, 1H), 7.26 (d, 1H), 6.92(d, 1H), 4.49 (s, 2H), 3.94 (m, 2H), 3.74 (m, 2H), 3.28 (m, 2H), 3.09(broad s, 2H).

To a suspension of NaH (60% dispersion, 1.14 g, 28.4 mmol) in dry THF(50 mL) under N₂ was added CH₃CN followed by 2-bromo-benzoic acid methylester (2 mL, 14.2 mmol). The reaction mixture was refluxed for 1.5 hour,then cooled to 0° C., quenched with water (1 mL), and concentrated invacuo. The residue was diluted with water and the aqueous layer wasextracted with hexane (2×), then acidified to pH 3-4 with 1 N aqueousHCl. The milky aqueous layer was extracted with CHCl₃ (3×), the combinedorganic layers were dried over sodium sulfate, filtered, andconcentrated. Purification on silica gel with 0-35% EtOAc in hexane aseluent provided 1.89 g (59% yield) of3-(2-bromo-phenyl)-3-oxo-propionitrile as a yellow oil. ¹H-NMR (d₆-DMSO)δ 11.8 (broad m, 1H, tautomers), 7.73 (broad s, 1H), 7.42 (m, 3H), 4.99(s, 0.3H, tautomer), 4.64 (s, 0.6H, tautomer); HPLC/MS m/z: 223.9, 225.9[MH]⁺.

To a solution of 3-(2-bromo-phenyl)-3-oxo-propionitrile (1.8 g, 8.03mmol) in absolute EtOH (25 mL) was added hydrazine hydrate (2.3 mL, 48.2mmol). The reaction mixture was refluxed for 23 hours, then cooled andpurified directly on silica gel with 0-10% MeOH in CH₂Cl₂ as eluent toprovide 1.33 g (70% yield) of 5-amino-3-(2-bromophenyl)pyrazole as asticky oil. ¹H-NMR (d₆-DMSO) δ: 11.7 (broad m, 1H, tautomers), 7.20-7.70(broad m, 4H), 5.76 (broad m, 1H), 5.03 (broad s, 1H), 4.60 (broad s,1H); HPLC/MS m/z: 238.0, 240.0 [MH]⁺.

To a solution of 5-amino-3-(2-bromophenyl)pyrazole (1.3 g, 5.46 mmol) inTHF (20 mL) was added dropwise benzoyl isothiocyanate (0.81 mL, 6.0mmol). The reaction mixture was stirred at room temperature for 3 hours,then 4 N aqueous solution of NaOH (4 mL) was added, and the reactionmixture was further stirred at 50° C. for 2 hours. The reaction mixturewas cooled to room temperature, neutralized to pH 7 with a saturatedsolution of NH₄Cl, and extracted with EtOAc (3×). The combined organiclayers were directly purified on silica gel with 0-10% MeOH in CH₂Cl₂ aseluent to provide 1.62 g (quant.) of[5-(2-bromo-phenyl)-2H-pyrazol-3-yl]-thiourea as a yellowish foam.¹H-NMR (d₆-DMSO) δ: 12.8 (broad s, 1H), 10.4 (broad s, 1H), 8.99 (broads, 1H), 8.52 (broad s, 1H), 7.76 (d, 1H), 7.50 (m, 2H), 7.36 (t, 1H),6.24 (broad s, 1H).

To a solution of [5-(2-bromo-phenyl)-2H-pyrazol-3-yl]-thiourea (1.6 g,5.38 mmol) in glacial AcOH (200 mL) was added a 1.5 M solution ofbromine in AcOH (3.59 mL, 5.38 mmol) dropwise under vigorous stirring.The resulting heterogeneous mixture was stirred at room temperature for2 hours then at 80° C. for 1 hour. The reaction was cooled to roomtemperature and concentrated to dryness. Water was added followed by 1 Naqueous NaOH to neutralize to pH 7. The resulting precipitate wasfiltered, washed with water and dried in vacuo. The solid was thenrefluxed in MeOH for 2 hours, filtered and washed with MeOH to give 588mg of pure 3-(2-bromo-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine as anoff-white solid. The filtrate was further purified on silica gel with0-10% MeOH in CH₂Cl₂ as eluent to provide another 460 mg of3-(2-bromo-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine for a total of1.408 g (88% yield). ¹H-NMR (d₆-DMSO) δ: 13.1 and 12.6 (2 broad s, 1H,NH+tautomer), 7.73 (d, 1H), 7.61 (broad m, 1H), 7.48 (m, 3H), 7.34 (t,1H); HPLC/MS m/z: 294.9, 296.9 [MH]⁺.

To a vial charged with3-(2-bromo-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine (30 mg, 0.135mmol), PS-DMAP (Argonaut resin, 0.54 g, 6 equiv.), and a stirring barwas added THF (1.5 mL) and 5-morpholin-4-ylmethyl-furan-2-carbonylchloride hydrochloride (180 mg, 0.675 mmol). The reaction mixture wasstirred at 70° C. for 17 hours, then cooled to room temperature andtreated with PS-trisamine (Argonaut resin, 0.76 g, 20 equiv.) at 50° C.for 8 hours. The resin was filtered, washed with DMF and the solvent wasevaporated. Purification on silica gel with 0-8% MeOH in CH₂Cl₂ aseluent provided 18 mg (27% yield) of5-morpholin-4-ylmethyl-furan-2-carboxylic acid[3-(2-bromo-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-yl]-amide as a whitesolid. ¹H-NMR (d₆-DMSO) δ: 13.7 and 13.5 (2 broad s, 1H, NH+tautomer),12.8 (broad s, 1H, NH), 7.65-7.82 (m, 3H), 7.49 and 7:55 (2 t, 1H,tautomers), 7.36 and 7.41 (2 t, 1H, tautomers), 6.58 (d, 1H), 3.59 (s,2H), 3.56 (t, 4H), 2.41 (broad s, 4H); HPLC/MS m/z: 488.0, 490.0 [MH]⁺.

Synthesis of cyclopropanecarboxylic acid[3-(4-hydroxy-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-yl]-amide

To a stirring suspension of3-(4-methoxyphenyl)-1H-pyrazolo[3,4-c]thiazol-5-ylamine (100 mg, 0.41mmol) in CH₂Cl₂ (2 mL) at −78° C. was added a 1 M solution of BBr₃ inCH₂Cl₂ (2.1 mL, 2.1 mmol) dropwise. The reaction mixture was slowlywarmed up to room temperature overnight, then it was quenched withwater, and the reaction mixture was neutralized to pH 6 with 1 N aqueousNaOH. The resulting precipitate was filtered, washed with water, anddried in vacuo to afford 70 mg (74% yield) of3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine as an off-whitesolid. ¹H-NMR (d₆-DMSO) δ: 9.8 (broad s, 1H), 7.57 (broad s, 2H), 7.42(d, 2H), 6.85 (d, 2H); HPLC/MS m/z: 233.0 [MH]⁺.

To a vial charged with3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine (60 mg, 0.259mmol), PS-DMAP (Argonaut resin, 1.05 g, 6 equiv.), and a stirring barwere added THF (2.5 mL) and cyclopropane-carbonyl chloride (118 μL, 1.29mmol). The reaction mixture was stirred at 70° C. for 15 hours, thencooled to room temperature and treated with PS-trisamine (Argonautresin, 1.26 g, 20 equiv.) at 50° C. for 48 hours. The resin wasfiltered, washed with DMF and the solvent was evaporated. Purificationon silica gel with 0-8% MeOH in CH₂Cl₂ as eluent provided 32 mg (41%yield) of cyclopropanecarboxylic acid[3-(4-hydroxy-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-yl]-amide as a whitesolid. ¹H-NMR (d₆-DMSO) δ: 13.3 (broad s, 1H), 12.6 (broad s, 1H), 9.80(broad s, 1H), 7.52 (d, 2H), 6.88 (d, 1H), 1.98 (m, 1H), 0.93 (m, 4H);HPLC/MS m/z: 301.0 [MH]⁺.

Synthesis of (S)-acetic acid1-[3-(2-chloro-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylcarbamoyl]-ethylester

To a solution of 3-(chlorophenyl)-3-oxo-propionitrile (4.0 g, 22.2 mmol)in absolute ethanol (21 mL) was added hydrazine hydrate (6.5 mL, 134mmol). The reaction mixture was stirred at 80° C. for 15 hours, then itwas concentrated in vacuo and purified on silica gel with 0-10% MeOH inCH₂Cl₂ as eluent to give 3.8 g (87% yield) of5-amino-3-(2-chlorophenyl)pyrazole as a yellow solid. ¹H NMR (d₆-DMSO)δ: 11.7 (broad s, 1H), 7.66 (d, 1H), 7.48 (d, 1H), 7.35 (t, 1H), 7.30(t, 1H), 5.83 (s, 1H), 4.84 (broad s, 2H); HPLC/MS m/z: 194.1 [MH]⁺.

To a solution of 5-amino-3-(2-chlorophenyl)pyrazole (3.8 g, 19.6 mmol)in THF (150 mL) was added dropwise benzoyl isothiocyanate (2.9 mL, 21.56mmol). The reaction mixture was stirred at room temperature for 4 hours,then 4 N aqueous solution of NaOH (8 mL) was added, and the reactionmixture was further stirred at room temperature for 17 hours. Thereaction mixture was neutralized to pH 7 with 1 N aqueous HCl, andextracted with EtOAc (3×). The combined organic layers were directlypurified on silica gel with 0-10% MeOH in CH₂Cl₂ as eluent to provide3.47 g (72% yield) of [542-chloro-phenyl)-2H-pyrazol-3-yl]-thiourea as atan solid.

To a solution of [5-(2-chloro-phenyl)-2H-pyrazol-3-yl]-thiourea (2.25 g,8.9 mmol) in glacial AcOH (900 mL) was added a 1.5 M solution of brominein AcOH (20 mL) dropwise under vigorous stirring. The resultingheterogeneous mixture was stirred at room temperature for 2 hours thenit was concentrated to dryness. Water was added followed by a saturatedsolution of aqueous sodium bicarbonate to neutralize to pH 7. Theresulting precipitate was filtered, washed with water and dried invacuo. Recrystallization from isopropyl alcohol afforded 1.48 g (66%yield) of 3-(2-chloro-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine as atan solid. ¹H-NMR (d₄-MeOH) δ: 7.64 (broad s, 1H), 7.53 (d, 1H), 7.42(m, 2H); HPLC/MS m/z: 251 [MH]⁺.

To a vial charged with3-(2-chloro-phenyl)-1H-pyrazolo[3,4-d]thiazol-5-ylamine (30 mg, 0.12mmol) and PS-DMAP (Argonaut resin, 450 mg, 5 equiv.) were added THF (0.8mL) and (S)-(−)-2-acetoxypropionyl chloride (126 mg, 0.84 mmol). Thereaction mixture was shaken at 70° C. overnight, then cooled to roomtemperature and treated with PS-trisamine (Argonaut resin, 1.03 g, 30equiv.) at 50° C. for 4 hours. The resin was filtered, washed with DMFand the solvent was evaporated. Purification by reverse-phasepreparative HPLC provided 21 mg (48% yield) of the title compound.¹H-NMR (d₆-DMSO) δ: 7.78 (broad m, 1H), 7.61 (broad m, 1H), 7.47 (broadm, 2H), 5.13 (q, 1H), 2.09 (s, 3H), 1.44 (d, 3H); HPLC/MS m/z: 365.0[MH]⁺.

Other Examples of Bicyclic Pyrazolo Kinase Modulators

Further examples of bicyclic pyrazolo kinase modulators produced by themethods provided herein are set forth in Tables 1, and 5-10.

TABLE 1

Other examples of bicyclic pyrazolo kinase modulators encompassed by thepresent invention are set forth in Tables 2 and 3 below.

TABLE 2

Q Q

TABLE 3

W W

Example 2 Bioassays

Kinase assays known to those of skill in the art may be used to assaythe inhibitory activities of the compounds and compositions of thepresent invention. Kinase assays include, but are not limited to, thefollowing examples.

For the purposes of these assays, the kinases are pre-diluted to a 10×working concentration in the following buffers. For Blk, CSK, and Lyn,the buffer composition is 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na₃VO₄,0.1% β-mercaptoethanol, and 1 mg/ml BSA. For Abl, CDK1, CDK5, Aurora-A,cSRC, Flt3, Fyn, GSK3α, GSK3β, Lck, Rsk1, Rsk2, Rsk3, and Yes, thebuffer composition is 20 mM MOPS, pH 7.0, 1 mM EDTA, 0.1%β-mercaptoethanol, 0.01% Brij-35, 5% glycerol, and 1 mg/ml BSA.

Kinases are assayed in final reaction volumes of 25 μl, comprising 5-10mU of kinase, the reaction is initiated by the addition of a MgATP mixof (final concentration) 10 mM MgAcetate and [γ³²P-ATP], having aspecific activity of approximately 500 cpm/pmol, concentration asrequired. The reaction is incubated for 40 minutes at room temperature,then stopped by adding 5 μl of a 3% phosphoric acid solution. 10 μl ofthe reaction mix is then spotted onto a Filtermat A filter and is thenwashed three times in 75 mM phosphoric acid for five minutes and once inmethanol. The filters are then dried and scintillation counted.Reactions further comprise reaction solutions and peptides, as presentedin Table 4 below.

TABLE 4 Kinase Reaction Solution Peptide Abl 8 mM MOPS, pH 7.0, 50 μMEAIYAAPFAKKK 0.2 mM EDTA Aurora-A 8 mM MOPS, pH 7.0, 200 μM LRRASLG(Kemptide) 0.2 mM EDTA Blk 50 mM Tris pH 7.5, 0.1 mg/ml poly (Glu, Tyr)4:1 0.1 mM EGTA, 0.1 mM Na₃VO₄, 0.1% β- mercaptoethanol CDK1 8 mM MOPS,pH 7.0, 0.1 mg/ml histone H1 0.2 mM EDTA CDK5 8 mM MOPS, pH 7.0, 0.1mg/ml histone H1 0.2 mM EDTA CSK 50 mM Tris pH 7.5, 0.1 mg/ml poly (Glu,Tyr) 4:1 0.1 mM EGTA, 0.1 mM Na₃VO₄, 0.1% β- mercaptoethanol cSRC 8 mMMOPS, pH 7.0, 250 μM KVEKIGEGTYGVVYK (Cdc2 peptide) 0.2 mM EDTA Flt3 8mM MOPS, pH 7.0, 50 μM EAIYAAPFAKKK 0.2 mM EDTA Fyn 50 mM Tris pH 7.5,250 μM KVEKIGEGTYGVVYK 0.1 mM EGTA, 0.1 mM (Cdc2 peptide) Na₃VO₄ GSK3α 8mM MOPS, pH 7.0, 20 μM 0.2 mM EDTA YRRAAVPPSPSLSRHSSPHQS(p)EDE EE(phospho GS2 peptide) GSK3β 8 mM MOPS, pH 7.0, 20 μM 0.2 mM EDTAYRRAAVPPSPSLSRHSSPHQS(p)EDE EE (phospho GS2 peptide) Lck 50 mM Tris pH7.5, 250 μM KVEKIGEGTYGVVYK (Cdc2 peptide) 0.1 mM EGTA, 0.1 mM Na₃VO₄Lyn 50 mM Tris pH 7.5, 0.1 mg/ml poly (Glu, Tyr) 4:1 0.1 mM EGTA, 0.1 mMNa₃VO₄, 0.1% β- mercaptoethanol Rsk1 8 mM MOPS, pH 7.0, 30 μM KKKNRTLSVA0.2 mM EDTA Rsk2 8 mM MOPS, pH 7.0, 30 μM KKKNRTLSVA 0.2 mM EDTA Rsk3 8mM MOPS, pH 7.0, 30 μM KKKNRTLSVA 0.2 mM EDTA Yes 8 mM MOPS, pH 7.0, 0.1mg/ml poly (Glu, Tyr) 4:1 0.2 mM EDTA

FLT3 Assay

Although this example presents the use of the FLT3 kinase domain, thekinase assays may use various forms of FLT3, including, for example, theentire molecule, the kinase domain, or a portion thereof.

Materials: Substrate peptide=Poly (Glu, Tyr) 4:1=poly EY (Sigma P-0275);βNADH (Sigma CAT#N-8129, FW=709.4); 2M MgCl₂; 1M HEPES buffer, pH 7.5;Phosphoenolpyruvate=PEP (Sigma CAT#P-7002, FW=234); Lactatedehydrogenase=LDH (Worthington Biochemical CAT#2756); Pyruvate Kinase=PK(Sigma CAT#P-9136); ATP (Sigma CAT#A-3377, FW=551); Greiner 384-well UVstar plate; and purified and autophosphorylated FLT3 kinase domain (FLT3KD).

Stock Solutions: 10 mM NADH (7.09 mg/mL in miliQH₂O) make fresh daily; 1mg/mL Poly EY (in miliQH₂O) store at −20° C.; 200 mM HEPES buffer, pH7.5 (10 ml 1M stock+40 ml miliQH₂O) supplemented with 1 mM DTT; 100 mMMgC12 (5 mL+95 ml dH₂O); 100 mM PEP (23.4 mg/mL in dH₂O) store at −20°C.; 10 mM ATP (5.51 mg/mL in dH₂O) store at −20° C. (dilute 1 mL intototal of 10 mL miliQH₂O daily=1 mM ATP working stock); 1000 U/mL PK(U/mg varies with lot) flash-freeze under liquid N2 and store at −80°C.; 1000 U/mL LDH (U/mg varies with lot) flash-freeze under liquid N2and store at −80° C.

Standard Assay Setup for 384-well format (500 reaction): 300 μM NADH; 10mM MgCl₂; 2 mM PEP; 45 U/mL PK; 60 U/mL LDH; 1 mg/mL Poly EY; 2.54 testcompound (in DMSO); 10 μg/mL autophosphorylated FLT3 kinase domain; 250μM ATP*; 100 mM HEPES buffer; QS with miliQ dH2O to 50 μL. Positivecontrols contained DMSO with no test compound. Negative controlscontained 5 μl of 0.5M EDTA (50 mM in the assay). The kinase reactionwas initiated at time t=0 by the addition of ATP.

The unphosphorylated FLT3 KD that was produced required pre-incubationwith MgATP to allow for autophosphorylation and full kinase activity.The autophosphorylation reaction was run in 100 mM HEPES buffer, 1 mMDTT, and 1 mg/mL FLT3 KD by the addition of ATP and MgCl₂ to a finalconcentration of 2 mM and 10 mM respectively. The reaction was allowedto proceed at room temperature for 90 minutes, and then was quenched bythe addition of EDTA to 50 mM. The autophosphorylated protein was thenaliquotted and flash frozen in liquid N₂ for use.

The activity was measured by following the time-dependent loss of NADHby absorbance spectroscopy at 340 nm. The linear portion of theresulting progress curve was then be analyzed by linear regression toget the activity in absorbance units/time, reported as the slope of thatbest fit line (moles/unit time can be calculated from using molarextinction coeffecient for NADH at 340 nm, 6250M⁻¹ cm⁻¹).

Data was analyzed using the equation: Z′=1−[3*(σ₊+σ⁻)/|μ₊−μ⁻|], where μdenotes the mean and σ the standard deviation. The subscript designatespositive or negative controls. The Z′ score for a robust screening assayshould be ≧0.50. The typical threshold=μ₊−3*σ₊. Any value that fallsbelow the threshold was designated a “hit.”

The dose response was measured using the equation:y=min+{(max−min)/(1+10^([compound]−log IC50))}, where y is the observedinitial slope, max is the slope in the absence of inhibitor, min is theslope at infinite inhibitor, and the IC₅₀ is the [compound] thatcorresponds to ½ the total observed amplitude (Amplitude=max−min). TheIC₅₀ is related to the K_(i) by the following equation:IC₅₀=K_(i)(1+[ATP]/Km).

To measure modulation, activation, or inhibition of FLT3 KD, a testcompound was added to the assay at a range of concentrations. Inhibitorsmay inhibit FLT3 KD activity at an IC₅₀ in the micromolar range, thenanomolar range, and, for example, in the subnanomolar range.

To measure the binding of a compound to FLT3 or FLT3 KD, a test kitmanufactured by Discoverx (Fremont, Calif.), ED-Staurosporine NSIP™Enzyme Binding Assay Kit (see U.S. Pat. No. 5,643,734) was used. Resultsare shown in Tables 5 and 6 below.

TABLE 5 Flt3 Structure IC50

A

B

A

B

B

B

In Table 5, A is <1 μM, B is 1-10 μM, and C is 11-100 μM.

TABLE 6 Structure FLT3 IC50

A

A

A

B

B

B

C

C

In Table 6, A is IC50<0.1 μM; B is 0.1 μM<IC50<1 μM; and C is IC50>1 μM.

Abl Assay

Although this example presents the use of the kinase domain of a mutantform of Abl T315I 0-P, the kinase assays may use various forms of mutantand wild type Abl, including, for example, the entire molecule, thekinase domain, or a portion thereof. The kinases used in the assays mayalso be of varying phosphorylation states. In the present example, amutant kinase at a zero phosphorylation state was used.

Materials: Abl substrate peptide=EAIYAAPFAKKK-OH (Biopeptide, SD CA);βNADH (Sigma CAT#N-8129, FW=709.4); 2M MgCl₂; 1M HEPES buffer, pH 7.5;Phosphoenolpyruvate=PEP (Sigma CAT#P-7002, FW=234); Lactatedehydrogenase=LDH (Worthington Biochemical CAT#2756); Pyruvate Kinase=PK(Sigma CAT#P-9136); ATP (Sigma CAT#A-3377, FW=551); Greiner 384-well UVstar plate; and Purified and unphosphorylated T315I Abl kinase domain(clone 5582d42PPt6p6).

Stock Solutions: 10 mM NADH (7.09 mg/mL in miliQH₂O) make fresh daily;10 mM Abl substrate peptide (13.4 mg/mL in miliQH₂O) store at −20° C.;100 mM HEPES buffer, pH 7.5 (5 mL 1M stock+45 mL miliQH₂O); 100 mM MgCl₂(5 mL 2M MgCl₂+95 ml dH₂O); 100 mM PEP (23.4 mg/mL in dH₂O) store at−20° C.; 10 mM ATP (5.51 mg/mL in dH₂O) store at −20° C. (dilute 50 μLinto total of 10 mL miliQH₂O daily=50 μM ATP working stock); 1000 U/mLPK (U/mg varies with lot) flash-freeze under liquid N2 and store at −80°C.; and 1000 U/mL LDH (U/mg varies with lot) flash-freeze under liquidN2 and store at −80° C.

Standard Assay Setup for 384-well format (50 μl reaction): 300 μM NADH;10 mM MgCl₂; 2 mM PEP; 45 U/mL PK; 60 U/mL LDH; 200 μM Abl substratepeptide; 2.5 μL test compound (in DMSO); 2 μg/mL Abl kinase domain; 10μM ATP; 100 mM HEPES buffer; QS with miliQ dH2O to 50 μL; Positivecontrols contained DMSO with no test compound. Negative controlscontained 5 μl of 0.5M EDTA (50 mM in the assay). The dephosphorylatedform of the c-Abl T315I mutant were used in the biochemical screeningassays. The kinase reaction was initiated at time t=0 by the addition ofATP.

The activity was measured by following the time-dependent loss of NADHby absorbance spectroscopy at 340 nm. The linear portion of theresulting progress curve was then be analyzed by linear regression toget the activity in absorbance units/time, reported as the slope of thatbest fit line (moles/unit time can be calculated from using molarextinction coefficient for NADH at 340 nm, 6250M⁻¹ cm⁻¹).

Data was analyzed using the equation: Z′=1−[3*(σ₊+σ⁻)/|μ₊−μ⁻|], where μdenotes the mean and σ the standard deviation. The subscript designatespositive or negative controls. The Z′ score for a robust screening assayshould be ≧0.50. The typical threshold=μ⁺−3*σ₊. Any value that fallsbelow the threshold was designated a “hit”.

Dose response was analyzed using the equation:y=min+{(max−min)/(1+10^([compound]−log IC50))}, where y is the observedinitial slope, max=the slope in the absence of inhibitor, min=the slopeat infinite inhibitor, and the IC₅₀ is the [compound] that correspondsto ½ the total observed amplitude (Amplitude=max−min).

To measure modulation, activation, or inhibition of AblKD, a testcompound was added to the assay at a range of concentrations. Inhibitorsmay inhibit AblKD activity at an IC₅₀ in the micromolar range, thenanomolar range, and, for example, in the subnanomolar range.

To measure the binding of a compound to Abl or AblKD, a test kitmanufactured by Discoverx (Fremont, Calif.), ED-Staurosporine NSIP™Enzyme Binding Assay Kit (see U.S. Pat. No. 5,643,734) was used. Resultsare presented in Tables 7 and 8 below.

TABLE 7 cAbl IC50 Structure (uM)

C

B

C

C

A

A

In Table 7, A is <1 μM, B is from 1-10 μM, and C is from 11-100 μM.

TABLE 8 Abl Abl T315I Y393F Compound IC50 IC50

A A

A A

A A

C C

B B

B C

C C

C C

In Table 8, A is IC50<0.5 μM; B is 0.5 μM<IC50<1 μM; C is IC50>1 μM.

MET and RON Assays

Although this example presents the use of wild type MET and RON M1243T,the kinase assays may use various forms of mutant and wild type RON orMET, including, for example, the entire intracellular domain, the kinasedomain, or a portion thereof. The kinases used in the assays may also beof varying phosphorylation states. In the case of RON, a mutant kinaseat a zero phosphorylation state was used.

For the MET and RON assays, the Kinase-Glo® assay system (Promega) wasused, which employs firefly luciferase to detect the amount of ATPfollowing a kinase reaction. This assay system has two steps. First, thekinase reaction is run for a designated period of time. Next, anequivalent volume of Kinase-Glo reagent is added to quench the kinasereaction and detect the ATP remaining in the sample. The total lightoutput is read by a plate-reading luminometer and the resulting signalis stable for 4 hours. Inhibition of kinase activity translates intolower ATP consumption and thus higher light output during the detectionstep.

Materials: Poly EY 4:1 (Sigma); MgCl₂ (2M stock available from LabSupport); HEPES buffer, pH 7.5; Bovine serum albumin (Roche 92423420);ATP (Sigma CAT#A-3377, FW=551); White Costar 384-well flat-bottom plate(VWR 29444-088); and SGX RON kinase clone (M1254T activation loopmutant) or SGX MET kinase clone.

MET enzyme mix: 100 mM HEPES pH 7.5, 10 mM MgCl₂, 0.3 mg/mL poly EY,0.1% BSA, and 0.4 μg/mL MET kinase.

RON enzyme mix: 100 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mg/mL poly EY, 0.1%BSA, and 3-5 μg/mL RON kinase (depending on enzyme prep).

To each well of a multiwell plate was added 15 μL of the enzyme mix, 1μL of compound at 20-times the desired final assay concentration (orDMSO for positive controls or 200 μM staurosporine for negativecontrols), and 4 μL of 50 μM ATP to start the reaction. The kinase wasreaction to allowed to proceed for 60 minutes at RT for the MET assay.For the RON assay, the reaction proceeded at RT for 60 minutes to 120minutes (depending on enzyme prep). 20 μL Kinase-Glo reagent was thenadded to each well. The reagent was incubated for at least 10 minutesbefore plate reading.

Data analysis was performed using methods similar to those describedabove in the Abl assay (Firepower®, Exegetix).

Results of the assay are presented in Table 9 below.

TABLE 9 Met Ron Compound IC50 IC50

A B

A B

A B

B B

B C

C C

C C

In Table 9, A is IC50<5 uM, B is 5 uM<IC50<20 uM, and C is IC50>20 uM.

Example 3 Preparation and Expression of Selected Kinases

Preparation of Human FLT3

FLT3 kinase domain for bioassays were prepared, for example, as follows.Human liver cDNA was synthesized using a standard cDNA synthesis kitfollowing the manufacturers' instructions. The template for the cDNAsynthesis was mRNA isolated from Hep G2 cells [ATCC HB-8065] using astandard RNA isolation kit. An open-reading frame for the FLT3 kinasedomain (FLT3 KD) was amplified from the human liver cDNA by thepolymerase chain reaction (PCR) using the following primers:

Forward primer: CACAAGTACAAAAAGCAATTTAGGTATG Reverse primer:CCGAATCTTCGACCTGAG.

The PCR product (840 base pairs expected) was electrophoresed on a 1.2%E-gel (Cat. #G5018-01, Invitrogen Corporation) and the appropriate sizeband was excised from the gel and eluted using a standard gel extractionkit. The eluted DNA was TOPO ligated into a GATEWAY™ (InvitrogenCorporation) adapted pcDNA6 AttB HisC vector which was custom TOPOadapted by Invitrogen Corporation. The resulting sequence of the geneafter being TOPO ligated into the vector, from the start sequencethrough the stop site was as follows: ATG GCC CTT 3′[FLT3KD]5′AA GGG CATCAT CAC CAT CAC CAC TGA. The FLT3 KD expressed using this vector had anN-terminal methionine, the kinase domain of FLT3 KD, and a C terminal6×His-tag.

Plasmids containing TOPO ligated inserts were transformed intochemically competent TOP 10 cells (Invitrogen Corporation,Cat.#C4040-10). Colonies were then screened for inserts in the correctorientation and small DNA amounts were purified using a “miniprep”procedure from 2 ml cultures, using a standard kit, following themanufacturer's instructions. For standard molecular biology protocolsfollowed here, see also, for example, the techniques described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 2001, and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,NY, 1989. The DNA that was in the “correct” orientation was thensequence verified.

A standard GATEWAY™ BP recombination was performed into pDONR201(Invitrogen Corporation, Cat.#11798014. Gateway technologyCat.#11821014) and the recombination reaction was transformed intochemically competent TOP 10 cells (Invitrogen Corporation,Cat.#C4040-10), and plated on selective media. One colony was pickedinto a miniprep and DNA was obtained (the “entry vector”).

The “entry vector” DNA was used in a standard GATEWAY™ LR recombinationwith pDEST8™ (Invitrogen Corporation, Cat.#11804010) and transformedinto chemically competent TOP 10 cells (Invitrogen Corporation,Cat.#C4040-10), and plated on selective media. One colony was pickedinto a miniprep and DNA was obtained (the “destination vector”).

The “destination vector” was then transformed into DH10 BAC chemicallycompetent cells (Invitrogen Corporation, Cat#10361012) which used sitespecific transposition to insert a foreign gene into a bacmid propogatedin E. coli. The transformation was then plated on selective media. 1-2colonies were picked into minipreps. The Nautilus Genomic miniprep kit(Active Motif, Cat.#50050) was used to purify the bacmid DNA. The bacmidwas then verified by PCR.

The plasmid was transfected and expressed in SF9 cells using thefollowing standard Bac to Bac protocol (Invitrogen Corporation,Cat.#10359-016).

Day 0: Seeded 9X10E5 cells per 35 mm well (of a 6 well plate) in 2 mlSf-900II SFM (Invitrogen Corporation, Cat. #10902-104) containing 1%Penicillin/Streptomycin (Invitrogen Corporation, Cat. # 15140122).Allowed cells to attach at 27° C. for 1 hour. In a Falcon 2059polypropylene 12×75 mm tube prepared the following solutions. Diluted 5μl of FLT3 KD miniprep bacmid DNA (Active Motif, Nautilus Genomic DNAMini Kit Cat. # 50050) into 100 μl Sf-900II SFM without pen/strep.Diluted 6 μl of CellFECTIN reagent (Invitrogen Corporation, Cat.#10362-010) into 100 μl A Sf-900II SFM without pen/strep. Combined the 2solutions together and incubated 30 minutes at room temperature. Washedthe cells once by aspirating old media and adding Sf-900II SFM withoutpen/strep. Removed media and added 0.8 ml Sf-900II SFM without pen/strepto each well. Added lipid/DNA to well. Incubated 5 hours in 27° C.incubator. Removed media and replaced with 2 ml Sf-900II SFM containingPenicillin/Streptomycin. Placed in 27° C. incubator.

Day 3, P1 to P2: In a T75 Tissue Culture Flask seeded 6X10E6 SF9 cellsin a total volume of 14 ml Sf-900II SFM containingPenicillin/Streptomycin. Allowed to attach for 1 hour. Using a 5 mlpipette, removed supernatant containing infectious P1 FLT3 KDBaculovirus particles from the transfected well of the 6 well andtransfered directly into T75 Flask. Placed in 27° C. incubator.

Day 10, P2 to P3: On Day 10 Harvested FLT3 KD Baculovirus supernatantand cells by vigorously pipetting the media to remove the cells from theflask wall. Pipetted the media and cells into a 15 ml sterile conicaltube and centrifuged the tube at @2000 rpm at room temperature for 5minutes. Saved supernatant (P2). Cells were analyzed for proteinexpression by western blot.

P3 infection: Seeded SF21 cells in a 500 ml suspension flask at 2X10E6cells per ml. in a total volume of 100 ml. Added infectious FLT3 KDsupernatant (14 ml) from P2 expression to suspension flask. Incubated at27 C, shaking at 120-130 rpm. Expressed protein for 72 hours.

Harvested 1 ml cells and western blotted to determine expression:Harvested P3 supernatant by centrifugation 3000 rpm for 15 minutes atroom temperature. Sterile filtered viral supernatant.

FLT3 KD Scale up: Seeded 6 liters of SF21 cells at 2X10E6 cells per mlin 1 liter of cells in 2-liter suspension flasks. Infected cells with 15ml of P3 FLT3 KD baculovirus per liter. Incubated at 27 C, shaking at120-130 rpm. Expressed protein for 48 hours. Harvested 1 ml cells fromeach liter and western blotted to determine expression. Remaining cellswere collected by centrifugation, and the pellets stored at −80° C.After thawing at room temperature, cells were lysed in cracking buffer(50 mM Tris-HCl, pH 8.0; 200 mM arginine; 150 mM NaCl; 10% glycerol;0.1% Igepal 630), and centrifuged to remove cell debris. The solublefraction was purified over an IMAC column charged with nickel(Pharmacia, Uppsala, Sweden), and eluted under native conditions with astep gradient of 400 mM imidazole in 50 mM Tris pH7.8, 10 mM methionine,10% glycerol. The FLT3 KD protein was then purified by gel filtrationusing a Superdex 200 preparative grade column equilibrated in GF4 buffer(10 mM HEPES, pH 7.5, 10 mM methionine, 500 mM NaCl, 5 mM DTT, and 10%glycerol). Fractions containing the purified FLT3 kinase domain werepooled and concentrated to 1-5 mg/ml.

Flt3, or a portion thereof, such as, for example, the kinase domain, mayalso be purified according to methods known to those of ordinary skillin the art. Examples of methods used to obtain Flt3 for assays, include,but are not limited to, those presented in Weisberg, Ellen, et al.,Cancer Cell 1:433-43, (2002).

Although the above protein kinase expression methods are exemplified bydescribing the expression of FLT3, other protein kinases (e.g. MET, andRON) were expressed using similar methodologies or methodologiesgenerally know in the art.

Preparation of Human Abl

A lambda phosphatase co-expression plasmid was constructed as follows.

An open-reading frame for Aurora kinase was amplified from a Homosapiens (human) HepG2 cDNA library (ATCC HB-8065) by the polymerasechain reaction (PCR) using the following primers:

Forward primer: TCAAAAAAGAGGCAGTGGGCTTTG Reverse primer:CTGAATTTGCTGTGATCCAGG.

The PCR product (795 base pairs expected) was gel purified as follows.The PCR product was electrophoresed on a 1% agarose gel in TAE bufferand the appropriate size band was excised from the gel and eluted usinga standard gel extraction kit. The eluted DNA was ligated for 5 minutesat room temperature with topoisomerase into pSB2-TOPO. The vectorpSB2-TOPO is a topoisomerase-activated, modified version of pET26b(Novagen, Madison, Wis.) wherein the following sequence has beeninserted into the NdeI site:CATAATGGGCCATCATCATCATCATCACGGTGGTCATATGTCCCTT and the followingsequence inserted into the BamHI site: AAGGGGGATCCTAAACTGCAGAGATCC. Thesequence of the resulting plasmid, from the Shine-Dalgarno sequencethrough the “original” NdeI site, the stop site and the “original” BamHIsite is as follows:AAGGAGGAGATATACATAATGGGCCATCATCATCATCATCACGGTGGTCATATGT CCCTT [ORF]AAGGGGGATCCTAAACTGCAGAGATCC. The Aurora kinase expressed using thisvector has 14 amino acids added to the N-terminal end(MetGlyHisHisHisHisHisHisGlyGlyHisMetSerLeu) and four amino acids addedto the C-terminal end (GluGlyGlySer).

The phosphatase co-expression plasmid was then created by inserting thephosphatase gene from lambda bacteriophage into the above plasmid(Matsui T, et al., Biochem. Biophys. Res. Commun., 2001, 284:798-807).The phosphatase gene was amplified using PCR from template lambdabacteriophage DNA (HinDIII digest, New England Biolabs) using thefollowing oligonucleotide primers:

Forward primer (PPfor): GCAGAGATCCGAATTCGAGCTCCGTCGACGGATGGAGTGAAAGAGATGCGC Reverse primer (PPrev):GGTGGTGGTGCTCGAGTGCGGCCGCAA GCTTTCATCATGCGCCTTCTCCCTGTAC.

The PCR product (744 base pairs expected) was gel purified. The purifiedDNA and non-co-expression plasmid DNA were then digested with Sad andXhoI restriction enzymes. Both the digested plasmid and PCR product werethen gel purified and ligated together for 8 hrs at 16° C. with T4 DNAligase and transformed into Top10 cells using standard procedures. Thepresence of the phosphatase gene in the co-expression plasmid wasconfirmed by sequencing. For standard molecular biology protocolsfollowed here, see also, for example, the techniques described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 2001, and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,NY, 1989.

The co-expression plasmid contains both the Aurora kinase and lambdaphosphatase genes under control of the lac promoter, each with its ownribosome binding site. By cloning the phosphatase into the middle of themultiple cloning site, downstream of the target gene, convenientrestriction sites are available for subcloning the phosphatase intoother plasmids. These sites include SacI SalI and EcoRI between thekinase and phosphatase and HinDIII, NotI and XhoI downstream of thephosphatase.

Protein Kinase Expression

An open-reading frame for c-Abl was amplified from a Mus musculus(mouse) cDNA library prepared from freshly harvested mouse liver using acommercially available kit (Invitrogen) by PCR using the followingprimers:

Forward primer: GACAAGTGGGAAATGGAGC Reverse primer: CGCCTCGTTTCCCCAGCTC.

The PCR product (846 base pairs expected) was purified from the PCRreaction mixture using a PCR cleanup kit (Qiagen). The purified DNA wasligated for 5 minutes at room temperature with topoisomerase intopSGX3-TOPO. The vector pSGX3-TOPO is a topoisomerase-activated, modifiedversion of pET26b (Novagen, Madison, Wis.) wherein the followingsequence has been inserted into the NdeI site: CATATGTCCCTT and thefollowing sequence inserted into the BamHI site:AAGGGCATCATCACCATCACCACTGATCC. The sequence of the resulting plasmid,from the Shine-Dalgarno sequence through the stop site and the BamHI,site is as follows: AAGGAGGA GATATACATATGTC CCTT[ORF]AAGGGCATCATCACCATCACCACTGATCC. The c-Abl expressed using this vector had threeamino acids added to its N-terminal end (Met Ser Leu) and 8 amino acidsadded to its C-terminal end (GluGlyHisHisHisHisHisHis).

A c-Abl/phosphatase co expression plasmid was then created by subcloningthe phosphatase from the Aurora co-expression plasmid of Example 1 intothe above plasmid. Both the Aurora co-expression plasmid and the Ablnon-co-expression plasmid were digested 3 hrs with restriction enzymesEcoRI and Nod. The DNA fragments were gel purified and the phosphatasegene from the Aurora plasmid was ligated with the digested c-Abl plasmidfor 8 hrs at 16° C. and transformed into Top10 cells. The presence ofthe phosphatase gene in the resulting construct was confirmed byrestriction digestion analysis.

This plasmid codes for c-Abl and lambda phosphatase co expression. Ithas the additional advantage of two unique restriction sites, XbaI andNdeI, upstream of the target gene that can be used for subcloning ofother target proteins into this phosphatase co-expressing plasmid.

The plasmid for Abl T315I was prepared by modifying the Abl plasmidusing the Quick Change mutagenesis kit (Stratagene) with themanufacturer's suggested procedure and the following oligonucleotides:

Mm05582dS4 5′-CCACCATTCTACATAATCATTGAGTTCATGACCTATGGG-3′ Mm05582dA45′-CCCATAGGTCATGAACTCAATGATTATGTAGAATGGTGG-3′.

Protein from the phosphatase co-expression plasmids was purified asfollows. The non-co-expression plasmid was transformed into chemicallycompetent BL21(DE3)Codon+RIL (Stratagene) cells and the co-expressionplasmid was transformed into BL21(DE3) pSA0145 (a strain that expressesthe lytic genes of lambda phage and lyses upon freezing and thawing(Crabtree S, Cronan J E Jr. J Bacteriol 1984 April; 158(1):354-6)) andplated onto petri dishes containing LB agar with kanamycin. Isolated,single colonies were grown to mid-log phase and stored at −80° C. in LBcontaining 15% glycerol. This glycerol stock was streaked on LB agarplates with kanamycin and a single colony was used to inoculate 10 mlcultures of LB with kanamycin and chloramphenicol, which was incubatedat 30° C. overnight with shaking. This culture was used to inoculate a 2L flask containing 500 mls of LB with kanamycin and chloramphenicol,which was grown to mid-log phase at 37° C. and induced by the additionof IPTG to 0.5 mM final concentration. After induction flasks wereincubated at 21° C. for 18 hrs with shaking.

The c-Abl T3151 KD was purified as follows. Cells were collected bycentrifugation, lysed in diluted cracking buffer (50 mM Tris HCl, pH7.5, 500 mM KCl, 0.1% Tween 20, 20 mM Imidazole, with sonication, andcentrifuged to remove cell debris. The soluble fraction was purifiedover an IMAC column charged with nickel (Pharmacia, Uppsala, Sweden),and eluted under native conditions with a gradient of 20 mM to 500 mMimidazole in 50 mM Tris, pH7.8, 500 mM NaCl, 10 mM methionine, 10%glycerol. The protein was then further purified by gel filtration usinga Superdex 75 preparative grade column equilibrated in GF5 buffer (10 mMHEPES, pH7.5, 10 mM methionine, 500 mM NaCl, 5 mM DTT, and 10%glycerol). Fractions containing the purified c-Abl T3151 KID kinasedomain were pooled. The protein obtained was 98% pure as judged byelectrophoresis on SDS polyacrylamide gels. Mass spectroscopic analysisof the purified protein showed that it was predominantly singlyphosphorylated. The protein was then dephosphorylated with ShrimpAlkaline Phosphatosase (MBI Fermentas, Burlington, Canada) under thefollowing conditions: 100U Shrimp Alkaline Phosphatase/mg of c-Abl T3151KD, 100 mM MgCl₂, and 250 mM additional NaCl. The reaction was runovernight at 23° C. The protein was determined to be unphosphorylated byMass spectroscopic analysis. Any precipitate was spun out and thesoluble fraction was separated from reactants by gel filtration using aSuperdex 75 preparative grade column equilibrated in GF4 buffer (10 mMHEPES, pH7.5, 10 mM methionine, 150 mM NaCl, 5 mM DTT, and 10%glycerol).

Example 4 Cell Assays

Flt3 cell line selection was performed according to the following twoknown methodologies: Kelly, et al, Cancer Cell 1:421-32 (2002); and Ye,et al, Blood 100: 2941-2949 (2002). MV4-11 and THP cells are maintainedin Iscove's Modified Dulbecco's Medium supplemented with 10% fetalbovine serum (FBS) and penicillin/streptomycin, Ba/F3 cells aremaintained in RPMI 1640 supplemented with 10% FBS,penicillin/streptomycin and 5 ng/ml recombinant mouse IL-3.

Cell Survival Assays

Compounds were tested in both of the following assays in triplicates.

96-well XTT assay: Cells are grown in their growth media containingvarious concentrations of compounds (triplicates) on a 96-well plate for72 hours at 37° C. The starting cell number is 8000 cells per well andvolume is 120 μl. At the end of the 72-hour incubation, 40 μl of XTTlabeling mixture (50:1 solution of sodium3′41-(pheylamino-carbonyl)-3,4-tetrazoliuml-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate and Electron-coupling reagent: PMS(N-methyl dibenzopyrazine methyl sulfate) is added to each well of theplate. After additional 2 hours of incubation at 37° C., the absorbancereading at 405 nm with background correction at 650 nm is done using aspectrophotometer.

384-well AlamarBlue assay: 90 μl of cell suspension is plated onto eachwell of a 384-well plate preprinted with 0.5 μl of compound in DMSO orDMSO only. The starting cell number is 4000 cells per well. After72-hour incubation, 10 μl of AlamarBlue solution (440 μM resazurin inPBS) is then added to each well of the plate. After additional 2-hourincubation at 37° C., fluorescence is measured using a TECAN platereading with excitation at 535 nm and emission 591 nm.

Results are shown in Table 10 below.

TABLE 10 MV4-11 (THP) Structure IC₅₀

B (C)

B (C)

B (C)

B (C)

In Table 10, A is <1 μM, B is 1-10 μM, and C is 11-100 μM.

Cellular Scratch Assay

Table 11 show the reagents that were used in the present cellularscratch assay.

TABLE 11 UNIT UNIT DESCRIPTION VENDOR CATALOG # SIZE PRICE Assay MediaOpti-MEM Invitrogen 11058-021 500 ml $24.20 NEAA, Invitrogen 11140-050100 ml $12.20 1% Pen-Strep Invitrogen 15140-122 100 ml $14.85 Sodiumpyruvate Invitrogen 11360-070 100 ml $6.60 Crystal Violet Sigma C3886 25 g $18.30 Accustain Formalin Sigma solution, 10% PBS HGF 30 ng/ul inH₂O Chemicon GF116 $195

MDCK, A549, A431, T24, H441 and SW579 cell lines were plated ontosix-well trays in triplicate for, +/−HGF and +/−compound, at densitiesthat would give confluent monolayers after 24 hours. Confluentmonolayers were incubated a further 48 hours to allow intercellularjunctions to mature before being serum-starved for 24 hours. A linearwound was generated on the monolayers by scraping with the tip of at 200μl pipette tip.

Unattached cells were washed off with PBS and assay media (Opti-MEM with1% Sodium Pyruvate, 1% Pen/Strep, 1% NEAA, +/−hepatocyte growth factor(“HGF”) at 90 ng/ml and +/−compound). After 24 hours, HGF was added tocells in the presence or absence of test compound. Cells were washed 1×with PBS then cells were fixed and stained with 10% formalin and 0.2%crystal violet in PBS for 10 minutes at room temperature. The wells arethen washed 5× with PBS adding 1 ml of PBS to each well, andphotographed. Photographs were inspected to determine whether the testcompound successfully reduced cell growth within the linear wound area.

The following compounds were shown to exhibit inhibitory properties inthe Cellular Scratch assay:

c-Met Phospho-ELISA Assay

Table 12 shows the reagents that were used in the present c-Metphosphor-ELISA assay.

TABLE 12 DESCRIPTION VENDOR CATALOG # UNIT SIZE Assay Media Opti-MEMInvitrogen 500 ml NEAA, Invitrogen 11140-050 100 ml 1% Pen-StrepInvitrogen 15140-122 100 ml Sodium pyruvate Invitrogen 11360-070 100 mlα-c-Met antibody Assay Design 905-076 100 μg 96w High binding Matrix4927 1 case Technologies Plate seals lysis buffer components Tris-ClpH7.4 (1M) NP-40 (10%) EDTA (500 mM) NaPP (100 mM) NaF (500 mM) NaCl(5M) Protease inhibitor, compete Sigma PMSF (100 mM in isopropanol)NaVO₄ (100 mM)* PBS, ice cold HGF 30 ng/ul in H₂O Chemicon GF116α-phosphotyrosine (4G10) Upstate cell 05-321 200 μg mention quote#2912in the notes to get signaling solutions special pricing Goat α-RabbitHRP Jackson 115-035-003 Immunoresearch BD OptEIA Reagent Set B BDBiosciences 550534 1 coating buffer (0.1M Na-carbonate, 250 ml pH 9.5)Assay Diluent 1000 ml Wash buffer (.05% Tween/PBS) 1000 ml (20x)SuperSignal ELISA Pico Pierce 37070 Chemiluminescent Substrate

Cells were plated at 3×10⁵ cell/ml in a total volume of 10 ml (3×10⁶total) and plated in a 100 mm dish in duplicate and incubated overnightat 37° C. with 5% CO₂. The day before the assay, the cells were washed1× with PBS and 10 mls of assay media (Opti-MEM I, reduced serum mediawith 1% NEAA, 1% Sodium Pyruvate, 1% Pen/Strep) was added to each plateof cells.

To appropriate wells of the plate was added 90 ng/ml of HGF followed bya 10 minute incubation at 37° C. under 5% CO₂. At the end of the 10minute incubation, lysis buffer was added to each well on the plate togive a final cell density of 2×10e4 per μl on ice. Cells were scrapedwith cell scraper, pipetted into an eppendorf tube, and incubated on icefor 15 minutes. Cells were centrifuged at 14,000 g for 5 minutes. 150 μlof lysate was pipetted into the wells on plate for serial dilutionacross the rows of the plate. To make the ELISA plate, commerciallyavailable rabbit Anti-Met antibodies were prepared at a concentration of0.125 μg/ml α-c-Met Ab in coating buffer (0.1M Na-carbonate, pH 9.5),and plated at 10 ml per plate (12.50 μl 100 μg/ml Ab/10 ml). In a highbinding multi-well plate, 100 μl Ab in coating buffer was added in eachwell, and each plate was covered with plate sealer and incubatedovernight at 4° C.

Excess antibody was removed and the ELISA plate was washed 4× with 200μl of wash buffer (0.05% Tween in PBS, pH 7.4). 150 μl of lysate wasadded per well and serially diluted across the rows of the plate. Plateswere sealed and incubated 2 hours at room temperature. The detectionantibody (α-p-Y 4G10, Upstate) was prepared in assay diluent. Theantibody was diluted 1:1000 (stock=2 μg/μl, 200 μg in 100 μl; f c.=2μg/ml) in assay diluent and 10 ml of diluted antibody per plate wasadded. The lysate was removed from the ELISA plates, and wells washedwith 200 μl per well wash buffer 4×. 100 μl of detection antibody wasadded to each well, covered, and incubated 1 hr at room temperature.Excess detection antibody was removed from the ELISA plates, and thewells washed with 200 μl per well with wash buffer 4×.

Secondary antibody, goat anti-rabbit HRP, was diluted 1:3000 in assaydiluent (3.33 μl per 10 mls diluent) and added at 10 ml of dilutedantibody per plate. Excess secondary antibody was removed from the ELISAplate, the plate was washed with 200 μl per well of wash buffer 4×.

Substrate Reagent A and Substrate Reagent B (Cat#37070 SuperSignal ELISAPico Chemiluminescent Substrate from Pierce) were added immediatelybefore use (10 ml resultant solution per plate). 100 μl substrate perwell was added, mixed for 1 minute and visualized with a luminometer.

1-26. (canceled)
 27. A method of modulating the activity of a proteinkinase comprising contacting said protein kinase with a compound ofclaim
 1. 28. A method of modulating the activity of a protein tyrosinekinase comprising contacting said protein tyrosine kinase with acompound of claim
 1. 29. A method of modulating the activity of areceptor tyrosine kinase comprising contacting said receptor tyrosinekinase with a compound of claim
 1. 30. A method of modulating theactivity of a protein kinase comprising contacting said protein kinasewith a compound of one of claims
 1. 31. A method for treating cancer ina human patient in need of such treatment, said method comprisingadministering to the patient a therapeutically effective amount of acompound of claim
 1. 32. (canceled)
 33. A compound having the formula:

wherein X is —O—, L¹ is —C(Z)—, or —SO₂—, wherein Z is ═O, ═S, or ═NR¹¹,wherein R¹¹ is hydrogen, —OH, cyano, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; andR¹ is hydrogen, —CF₃, amino, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl,—OR¹², or —NR¹³R¹⁴, wherein R¹², R¹³, and R¹⁴ are independently selectedfrom hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, R¹³ andR¹⁴ are optionally joined to from a ring with the nitrogen to which theyare attached, wherein said ring is substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R² issubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl, wherein R² is attached to theremainder of the molecule via a carbon atom to form a carbon-carbonbond, or a pharmaceutically acceptable salt thereof.
 34. The compound ofclaim 33, wherein L¹ is —C(Z)—, wherein Z is ═O.
 35. The compound ofclaim 33, wherein R² is substituted or unsubstituted C₁-C₂₀ alkyl,substituted or unsubstituted 2 to 20 membered heteroalkyl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 3 to 8membered heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.
 36. The compound of claim 33,wherein R² is substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted 2 to 10 membered heteroalkyl, substituted or unsubstitutedC₃-C₇ cycloalkyl, substituted or unsubstituted 3 to 7 memberedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.
 37. The compound of claim 33, wherein R² is(1) unsubstituted C₁-C₁₀ alkyl; (2) unsubstituted 2 to 10 memberedheteroalkyl; (3) unsubstituted C₃-C₇ cycloalkyl; (4) unsubstituted 3 to7 membered heterocycloalkyl; (5) unsubstituted aryl; (6) unsubstitutedheteroaryl; (7) substituted C₁-C₁₀ alkyl; (8) substituted 2 to 10membered heteroalkyl; (9) substituted C₃-C₇ cycloalkyl; (10) substituted3 to 7 membered heterocycloalkyl; (11) substituted aryl; or (12)substituted heteroaryl; wherein (7), (8), (9), or (10) is substitutedwith an oxo, —OH, —CF₃, —COOH, halogen, R²¹-substituted or unsubstitutedC₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl, -L²²-C(O)R³, -L²²-OR⁴, -L²²-NR⁴R⁵, OR⁴, or-L²²-S(O)_(m)R⁶, (11) or (12) is substituted with an —OH, —CF₃, —COOH,halogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²-substitutedor unsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl,R²²-substituted or unsubstituted heteroaryl, -L²²-C(O)R³, -L²²-OR⁴,-L²²-NR⁴R⁵, or -L²²S(O)_(m)R⁶, R³ is hydrogen, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, R²²-substituted or unsubstitutedheteroaryl, —OR³¹, or —NR³²R³³, wherein R³¹, R³², and R³³ areindependently hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl,R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, or R²²-substituted or unsubstituted heteroaryl, R⁴and R⁵ are independently hydrogen, —CF₃, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, R²²-substituted or unsubstitutedheteroaryl, or —C(O)R⁴¹, wherein R⁴¹ is hydrogen, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl, R⁶ is hydrogen, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl, L²² is a bond, unsubstituted C₁-C₁₀ alkyleneor unsubstituted heteroalkylene, m is 0, 1, or 2, R²¹ is oxo, —OH,—COOH, —CF₃, amino, halogen, R²³-substituted or unsubstituted 2 to 10membered heteroalkyl, R²³-substituted or unsubstituted C₃-C₇ cycloalkyl,R²³-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²⁴-substituted or unsubstituted aryl, or R²⁴-substituted orunsubstituted heteroaryl, R²² is —OH, —COOH, amino, halogen, —CF₃,R²³-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²³-substituted or unsubstituted C₃-C₇ cycloalkyl, R²³-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²⁴-substituted orunsubstituted aryl, or R²⁴-substituted or unsubstituted heteroaryl, R²³is oxo, —OH, —COOH, amino, halogen, —CF₃, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl, and R²⁴ is —OH, —COOH,amino, halogen, —CF₃, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to7 membered heterocycloalkyl, unsubstituted aryl, unsubstitutedheteroaryl.
 38. The compound of claim 37, wherein if R²¹ or R²² issubstituted or unsubstituted heterocycloalkyl, the heterocycloalkyl ishydantoinyl, dioxolanyl, dioxanyl, trioxanyl, tetrahydrothienyl,tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl,tetrahydrothiopyranyl, pyrrolidinyl, morpholino, piperidinyl, orpiperazinyl.
 39. The compound of claim 37, wherein if R²¹ or R²² issubstituted or unsubstituted heteroaryl, then the heteroaryl ispyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl,pyrrolyl, pyridyl, pyrazyl, pyrimidyl, pyridazinyl, thiazolyl,isothioazolyl, triazolyl, thienyl, triazinyl, or thiadiazolyl.
 40. Thecompound of claim 37, wherein R² is substituted or unsubstituted aryl,or substituted or unsubstituted heteroaryl.
 41. The compound of claim37, wherein R² is substituted or unsubstituted cycloalkyl, substitutedor unsubstituted heterocycloalkyl, substituted or unsubstituted alkyl,or substituted or unsubstituted heteroalkyl.
 42. The compound of claim40, wherein the R² is substituted with a(n) (1) unsubstituted C₁-C₁₀alkyl, (2) unsubstituted 2 to 10 membered heteroalkyl, (3) unsubstitutedC₃-C₇ cycloalkyl, (4) unsubstituted 3 to 7 membered heterocycloalkyl,(5) unsubstituted aryl, (6) unsubstituted heteroaryl, (7) halogen, (8)—OH, (9) amino, (10) —CF₃, (11) 3 to 7 membered heterocycloalkylsubstituted with unsubstituted C₁-C₁₀ alkyl, or (12) C₁-C₁₀ alkylsubstituted with an unsubstituted aryl.
 43. The compound of claim 40,wherein the R² is substituted with a (1) halogen, (2) -L²²-C(O)R³, (3)-L²²-OR⁴, (4) -L²²-NR⁴R⁵, or (5) -L²²-S(O)_(m)R⁶.
 44. The compound ofclaim 40, wherein the R² substituent is (1) -L²²-C(O)R³, wherein R³ ishydrogen, unsubstituted C₁-C₁₀ alkyl, —OR³¹, —NR³²R³³, wherein R³¹, R³²,and R³³ are independently hydrogen, unsubstituted C₁-C₁₀ alkyl, orunsubstituted C₃-C₇ cycloalkyl, and L²² is unsubstituted C₁-C₁₀alkylene, (2) -L²²-OR⁴, wherein R⁴ is hydrogen, —CF₃, —CHF₂,unsubstituted C₁-C₁₀ alkyl, unsubstituted C₃-C₇ cycloalkyl,unsubstituted C₁-C₁₀ cycloalkylalkyl, or —C(O)R⁴¹, wherein R⁴¹ ishydrogen, or unsubstituted C₁-C₁₀ alkyl, (3) -L²²-NR⁴R⁵, wherein R⁴ andR⁵ are independently hydrogen, unsubstituted C₁-C₁₀ alkyl, or —C(O)R⁴¹,wherein R⁴¹ is independently hydrogen, or unsubstituted C₁-C₁₀ alkyl, or(4) L²²S(O)_(m)R⁶, wherein R⁷ is hydrogen or unsubstituted C₁-C₁₀ alkyl.45. The compound of claim 37, wherein if R² is substituted orunsubstituted aryl, then the aryl is phenyl, benz[cd]indol-2(1H)-one-yl,oxindolyl, indazolinonyl, benzoimidazolyl, indolyl, benzodioxanyl,cournarinyl, chromonyl, benzopyrazyl, naphthyl, quinolyl, orisoquinolyl; if R² is substituted or unsubstituted heteroaryl, then theheteroaryl is pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl,oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyrazyl, pyridazinyl, thiazolyl,isothioazolyl, triazolyl, thienyl, triazinyl, thiadiazolyl, or if R² issubstituted or unsubstituted heterocycloalkyl, then the heterocycloalkylis hydantoinyl, dioxolanyl, dioxanyl, trioxanyl, tetrahydrothienyl,tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl,tetrahydrothiopyranyl, pyrrolidinyl, morpholino, piperidinyl, orpiperazinyl.
 46. The compound of claim 37, wherein if (11) or (12) issubstituted with a R²¹-substituted or unsubstituted 3 to 7 memberedheterocycloalkyl, then the heterocycloalkyl is dioxolanyl, dioxanyl,trioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl,tetrahydrothiopyranyl, pyrrolidinyl, morpholino, piperidinyl, orpiperazinyl; or if (11) or (12) is substituted with a R²²-substituted orunsubstituted heteroaryl, then the heteroaryl is pyrazolyl, furanyl,imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl,pyrazyl, pyrimidyl, pyridazinyl, thiazolyl, isothioazolyl, triazolyl, orthienyl, triazinyl, or thiadiazolyl.
 47. The compound of claim 33,wherein R¹ is hydrogen, amino, substituted or unsubstituted C₁-C₁₀alkyl, substituted or unsubstituted 2 to 10 membered heteroalkyl,substituted or unsubstituted C₃-C₇ cycloalkyl, substituted orunsubstituted 3 to 7 membered heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.
 48. Thecompound of claim 33, wherein R¹ is (1) —CF₃; (2) unsubstituted C₁-C₁₀alkyl; (3) unsubstituted 2 to 10 membered heteroalkyl; (4) unsubstitutedC₃-C₇ cycloalkyl; (5) unsubstituted 3 to 7 membered heterocycloalkyl;(6) unsubstituted aryl; (7) unsubstituted heteroaryl; (8) substitutedC₁-C₁₀ alkyl; (9) substituted 2 to 10 membered heteroalkyl; (10)substituted C₃-C₇ cycloalkyl; (11) substituted 3 to 7 memberedheterocycloalkyl; (12) substituted aryl; or (13) substituted heteroaryl;wherein (8), (9), (10) or (11) is substituted with an oxo, —OH, —CF₃,—COOH, halogen, R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl,R¹⁵-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹⁵-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹⁶-substituted orunsubstituted aryl, R¹⁶-substituted or unsubstituted heteroaryl,-L¹¹-C(O)R¹⁰⁰, -L¹¹-OR¹⁰⁴, -L¹¹-NR¹⁰⁴R¹⁰⁵, or -L¹¹-S(O)_(q)R¹⁰⁷, (12) or(13) is substituted with an —OH, —CF₃. —COOH, halogen, R¹⁵-substitutedor unsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹⁵-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R16-substituted or unsubstituted aryl, R¹⁶-substituted or unsubstitutedheteroaryl, -L¹¹-C(O)R¹⁰⁰, -L¹¹-OR¹⁰⁴, -L¹¹-NR¹⁰⁴R¹⁰⁵, or-L¹¹-S(O)_(q)—R¹⁰⁷, R¹⁰⁰ is hydrogen, R¹⁵-substituted or unsubstitutedC₁-C₁₀ alkyl, R¹⁵-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹⁵-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹⁶-substituted or unsubstituted aryl, R¹⁶-substituted or unsubstitutedheteroaryl, —OR¹⁰¹, or —NR¹⁰²R¹⁰³, wherein R¹⁰¹, R¹⁰², R¹⁰³ areindependently hydrogen, R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl,R¹⁵-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹⁵-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹⁶-substituted orunsubstituted aryl, or R¹⁶-substituted or unsubstituted heteroaryl, R¹⁰⁴and R¹⁰⁵ are independently hydrogen, —CF₃, R¹⁵-substituted orunsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹⁵-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹⁵-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹⁶-substituted or unsubstituted aryl, R¹⁶-substituted or unsubstitutedheteroaryl, or —C(O)R¹⁰⁶, wherein R¹⁰⁶ is independently hydrogen,R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹⁵-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹⁵-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, orR¹⁶-substituted or unsubstituted heteroaryl, R¹⁰⁷ is hydrogen,R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl, R¹⁵-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹⁵-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹⁵-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, orR¹⁶-substituted or unsubstituted heteroaryl, L¹¹ is a bond,unsubstituted C₁-C₁₀ alkylene, or unsubstituted heteroalkylene, q is 0,1, or 2, R¹⁵ is oxo, —OH, —COOH, —CF₃, halogen, R¹⁷-substituted orunsubstituted C₁-C₁₀ alkyl, R¹⁷-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹⁷-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹⁷-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹⁸-substituted or unsubstituted aryl, or R¹⁸-substituted orunsubstituted heteroaryl, R¹⁶ is —OH, —COOH, —CF₃, halogen,R¹⁷-substituted or unsubstituted C₁-C₁₀ alkyl, R¹⁷-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹⁷-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹⁷-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹⁸-substituted or unsubstituted aryl, orR¹⁸-substituted or unsubstituted heteroaryl, R¹⁷ is oxo, —OH, —COOH,—CF₃, halogen, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to7 membered heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl, and R¹⁸ is —OH, —COOH, —CF₃, halogen, unsubstituted C₁-C₁₀alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.
 49. The compound ofclaim 48, wherein if R¹⁵ or R¹⁶ is substituted or unsubstitutedheterocycloalkyl, the heterocycloalkyl is hydantoinyl, dioxolanyl,dioxanyl, trioxanyl, tetrahydrothienyl, tetrahydrofuranyl,tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl,pyrrolidinyl, morpholino, piperidinyl, or piperazinyl.
 50. The compoundof claim 48, wherein if R¹⁵ or R¹⁶ is substituted or unsubstitutedheteroaryl, the heteroaryl is pyrazolyl, furanyl, imidazolyl,isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrazyl,pyrimidyl, pyridazinyl, thiazolyl, isothioazolyl, triazolyl, or thienyl,triazinyl, or thiadiazolyl.
 51. The compound of claim 48, wherein R¹⁵ isoxo, —OH, —COOH, —CF₃, halogen, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl, and R¹⁶ is —OH, —COOH,—CF₃, halogen, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to7 membered heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl.
 52. The compound of claim 33, wherein R¹ is (1)unsubstituted C₁-C₁₀ alkyl; (2) unsubstituted 2 to 10 memberedheteroalkyl; (3) unsubstituted C₃-C₇ cycloalkyl; (4) unsubstituted 3 to7 membered heterocycloalkyl; (5) substituted C₁-C₁₀ alkyl; (6)substituted 2 to 10 membered heteroalkyl; (7) substituted C₃-C₇cycloalkyl; (8) substituted 3 to 7 membered heterocycloalkyl; (9)substituted phenyl; or (10) substituted heteroaryl; wherein (5), (6),(7), or (8) is substituted with an oxo, —OH, —CF₃. —COOH, halogen,R¹⁵-substituted or unsubstituted C₁-C₁₀ alkyl, or R¹⁵-substituted orunsubstituted 2 to 10 membered heteroalkyl, and wherein (9) or (10) issubstituted with an —OH, —CF₃. —COOH, halogen, R¹⁵-substituted orunsubstituted C₁-C₁₀ alkyl, or R¹⁵-substituted or unsubstituted 2 to 10membered heteroalkyl.
 53. The compound of claim 52, wherein R¹⁵ is oxo,—OH, —COOH, —CF₃, halogen, unsubstituted C₁-C₁₀ alkyl, or unsubstituted2 to 10 membered heteroalkyl.
 54. The compound of claim 33, wherein R¹is (1) unsubstituted C₁-C₁₀ alkyl, (2) unsubstituted 2 to 10 memberedheteroalkyl, (3) unsubstituted C₃-C₇ cycloalkyl, (4) unsubstituted 3 to7 membered heterocycloalkyl; (5) C₁-C₁₀ alkyl substituted with an oxo,—OH, —COOH, —CF₃, halogen, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2to 10 membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl,unsubstituted 3 to 7 membered heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl; (6) 2 to 10 membered heteroalkyl substitutedwith an oxo, —OH, —CF₃, —COOH, halogen, unsubstituted C₁-C₁₀ alkyl, 2 to10 membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3to 7 membered heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl; (7) C₃-C₇ cycloalkyl substituted with —OH, —CF₃, —COOH,halogen, unsubstituted C₁-C₁₀ alkyl, or unsubstituted 2 to 10 memberedheteroalkyl; (8) 3 to 7 membered heterocycloalkyl substituted with —OH,—CF₃, —COOH, halogen, unsubstituted C₁-C₁₀ alkyl, or unsubstituted 2 to10 membered heteroalkyl; (9) phenyl substituted with —OH, —CF₃, —COOH,halogen, unsubstituted C₁-C₁₀ alkyl, or unsubstituted 2 to 10 memberedheteroalkyl; or (10) heteroaryl substituted with —OH, —CF₃, —COOH,halogen, unsubstituted C₁-C₁₀ alkyl, or unsubstituted 2 to 10 memberedheteroalkyl.
 55. The compound of claim 48, wherein if R¹ is asubstituted or unsubstituted heteroaryl, the heteroaryl is pyrazolyl,furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl,pyridyl, pyrimidyl, pyrazyl, pyridazinyl, thiazolyl, isothioazolyl,triazolyl, thienyl, triazinyl, or thiadiazolyl.
 56. The compound ofclaim 48, wherein if R¹ is a substituted or unsubstitutedheterocycloalkyl, then the heterocycloalkyl is hydantoinyl, dioxolanyl,dioxanyl, trioxanyl, tetrahydrothienyl, tetrahydrofuranyl,tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl,pyrrolidinyl, morpholino, piperidinyl, or piperazinyl.
 57. The compoundof claim 48, wherein the R¹ is substituted with a (1) halogen, (2)-L¹¹-C(O)R¹⁰⁰, (3) -L¹¹-OR¹⁰⁴, (4) -L¹¹-OR¹⁰⁴, (5) -L¹¹-S(O)_(q)R¹⁰⁷.58. The compound of claim 57, wherein R¹⁵ is oxo, —OH, —COOH, —CF₃,halogen, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10 memberedheteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to 7membered heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl, and R¹⁶ is —OH, —COOH, —CF₃, halogen, unsubstituted C₁-C₁₀alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.
 59. A pharmaceuticalcomposition comprising a compound of claim 33 in admixture with apharmaceutically acceptable excipient.